Methods of treating dementia  associated with alzheimer&#39;s disease with protective protein/cathepsin a (ppca)

ABSTRACT

Methods are provided for the prognosis, diagnosis and treatment of various pathological states, including cancer, chemotherapy resistance and dementia associated with Alzheimer&#39;s disease. The methods provided herein are based on the discovery that various proteins with a high level of sialylation are shown herein to be associated with disease states, such as, cancer, chemotherapy resistance and dementia associated with Alzheimer&#39;s disease. Such methods provide a lysosomal exocytosis activity profile comprising one or more values representing lysosomal exocytosis activity. Also provided herein, is the discovery that low lysosomal sialidase activity is associated with various pathological states. Thus, the methods also provide a lysosomal sialidase activity profile, comprising one or more values representing lysosomal sialidase activity. A lysosomal sialidase activity profile is one example of a lysosomal exocytosis activity profile.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/805,075, filed Nov. 9, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/187,349, filed Jun. 20, 2016, issued as U.S.Pat. No. 9,840,727 on Dec. 12, 2017, which is a continuation of U.S.patent application Ser. No. 14/239,728, filed Feb. 19, 2014, issued asU.S. Pat. No. 9,399,791 on Jul. 26, 2016, which is a National StageApplication of PCT/US2012/052629, filed Aug. 28, 2012, which claims thebenefit of U.S. Provisional Application Ser. No. 61/544,855, filed Oct.7, 2011, and U.S. Provisional Application Ser. No. 61/529,675, filedAug. 31, 2011. Each of these priority applications is incorporatedherein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Federal Government support under GM060950awarded by the National Institutes of Health. The United StatesGovernment has certain rights in the invention. This invention was alsosupported by the American Lebanese Syrian Associated Charities (ALSAC)of St. Jude Children's Research Hospital.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology, cancerand Alzheimer's disease therapeutics and diagnostics.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of S88435_1110US_C3_Seq_List.txt, a creation date of Nov. 26, 2019,and a size of 12 KB. The sequence listing filed via EFS-Web is part ofthe specification and is hereby incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

The prognosis of a disease or pathological condition in a subject can begreatly improved with an early diagnosis. However, reliable prognosticand diagnostic methods are lacking for managing disease states. Forexample, for Alzheimer's disease, the only definitive diagnostic test isto determine whether amyloid plaques are present in a subject's braintissue, a determination that can only be made after death. Thus, due tothe lack of suitable diagnostic methods only a tentative diagnosis canbe provided. In another example, diagnosis and prognosis of a cancer areimportant for choosing the best treatment options in order to improveoutcome. There is also a need for diagnostic and prognostic tests topredict the efficacy of a particular chemotherapy regime to determinethe best treatment options for a subject.

Therefore, there is a significant need in the art for more accurate andreliable diagnostic and prognostic methods for cancer and Alzheimer'sdisease.

BRIEF SUMMARY OF THE INVENTION

Methods are provided for the prognosis, diagnosis and treatment ofvarious pathological states, including cancer, chemotherapy resistanceand dementia associated with Alzheimer's disease. The methods providedherein are based on the discovery that various proteins with a highlevel of sialylation are shown herein to be associated with diseasestates, such as, cancer, chemotherapy resistance and dementia associatedwith Alzheimer's disease. Such methods provide a lysosomal exocytosisactivity profile comprising one or more values representing lysosomalexocytosis activity. Also provided herein, is the discovery that lowlysosomal sialidase activity is associated with various pathologicalstates. Thus, the methods also provide a lysosomal sialidase activityprofile, comprising one or more values representing lysosomal sialidaseactivity. A lysosomal sialidase activity profile is one example of alysosomal exocytosis activity profile. As such, the level of lysosomalexocytosis activity and/or lysosomal sialidase activity is predictive ofa diagnosis and/or prognosis of cancer, chemotherapy resistance ordementia associated with Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays a summary model of the role of NEU1 in cancer.

FIG. 2 depicts the presence of lysosomal proteins in the CSF and thecorrelation of these proteins with Alzheimer's disease.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Overview

Provided herein are methods for the diagnosis and prognosis of variouspathological states by looking at the lysosomal exocytosis activity in asample. The level of lysosomal exocytosis activity can serve as a markerfor the diagnosis and/or prognosis of pathological conditions including,for example, cancer, chemotherapy resistance and dementia associatedwith Alzheimer's disease. Various activity profiles are provided hereinfor the diagnosis and/or prognosis of cancer, chemotherapy resistance,and dementia associated with Alzheimer's disease.

II. Types of Profiles

As used herein, a “profile” comprises one or more values correspondingto a measurement of a marker(s) representing an activity in a sample.Various profiles are disclosed herein which can be used for theprognosis and/or diagnosis of a given pathological state. Such profilesinclude: a lysosomal exocytosis activity profile, a sialylation activityprofile, a lysosomal sialidase activity profile, a NEU1 substratesialylation activity profile and a NEU1 level activity profile. Each ofthese profiles is explained in detail herein and summarized in Table 1herewith.

By “lysosomal exocytosis activity profile” is meant a profile of one ormore values representing lysosomal exocytosis activity. As used herein,“lysosomal exocytosis activity” is meant a measure of the level ofexocytosis in a sample. Various markers can be used to determine thelysosomal exocytosis activity of a sample. Such markers include one ormore of the following: (1) the level of NEU1 protein or direct enzymaticactivity of NEU1; (2) the protein level of one or more NEU1 substrates;(3) the protein level of one or more lysosomal proteins; (4) the proteinlevel of one or more lysosomal proteases; (5) the protein level ofLAMP-1; (6) the protein level of hexosaminidase beta; (7) the proteinlevel of mannosidase alpha; or (8) the protein level of one or morecathepsins; (9) any marker for a sialylation activity profile providedherein; or (10) any marker for a lysosomal sialidase activity profileprovided herein. Once the level of each of a given marker is determined,it becomes a value in the lysosomal exocytosis activity profile. Thelysosomal exocytosis activity profile can comprise 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or more lysosomal exocytosis activityvalues of the various lysosomal exocytosis activity markers providedherein.

In one embodiment, one type of lysosomal exocytosis activity profile isa sialylation activity profile. By “sialylation activity profile” ismeant a profile of one or more values representing sialylation activity.As used herein, “sialylation activity” is meant a measure of thesialylation level of a population of proteins in a sample or thesialylation level of one or more proteins in a sample. Various markerscan be used to determine sialylation activity. Such markers include oneor more of the following: (1) the overall level of sialylation in asample; (2) the level of NEU1 protein or direct enzymatic activity ofNEU1; (3) the level of sialylation of one or more NEU1 substrates; (4)the protein level of one or more NEU1 substrates; or (5) any marker fora lysosomal sialidase activity profile, as discussed in further detailelsewhere herein or outlined in Table 1. Once the level of each of agiven marker is determined, it becomes a value in the sialylationactivity profile. The sialylation activity profile can comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more sialylation activityvalues of the various sialylation activity markers provided herein.

In one embodiment, one type of sialylation activity profile is alysosomal sialidase activity profile. By “lysosomal sialidase activityprofile” is meant a profile of one or more values representing lysosomalsialidase activity. By “lysosomal sialidase activity” is meant a director indirect measure of lysosomal sialidase activity. Various markers canbe used to determine lysosomal sialidase activity in a sample. Thevarious markers representing the lysosomal sialidase activity in asample include any one or more of the following: (1) the level of NEU1protein or the level of direct enzymatic activity of NEU1; (2) theprotein level of one or more NEU1 substrate; (3) the level ofsialylation of one or more NEU1 substrate; or (4) the activity level ofone or more NEU1 substrate. Once the level or activity of a given markeris determined, it becomes a value in the lysosomal sialidase activityprofile. Thus, the lysosomal sialidase activity profile can comprise anycombination of the lysosomal sialidase activity markers provided herein.The lysosomal sialidase activity profile can comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 or more lysosomal sialidase activityvalues of the various lysosomal sialidase activity markers providedherein.

In one embodiment, one type of lysosomal sialidase activity profile is aNEU1 substrate sialylation activity profile. By “NEU1 substratesialylation activity profile” is meant measuring lysosomal sialidaseactivity in a sample by determining the level of sialylation of one ormore NEU1 substrates. The various markers representing lysosomalsialidase activity that are encompassed in a NEU1 substrate sialylationactivity profile include: (1) the level of sialylation of one or moreNEU1 substrate; (2) the level of sialylation of LAMP-1; (3) The level ofsialylation of MUC-1; or (4) the level of sialylation of NEU1 and MUC-1.The NEU1 substrate sialylation activity profile can comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more marker values provided bythe sialylation level of the various NEU1 substrates.

In another embodiment, one type of lysosomal sialidase activity profileis a NEU1 level activity profile. By “NEU1 level activity profile” ismeant measuring lysosomal sialidase activity in a sample by determiningthe protein level of any non-MUC-1 NEU1 substrate or of NEU1 itself. Thevarious markers representing lysosomal sialidase activity that areencompassed in a NEU1 level activity profile include: (1) the level ofNEU1 protein; (2) the protein level of any one or more non-MUC-1 NEU1substrate; or (3) the protein level of LAMP-1. The NEU1 level activityprofile can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or more marker values provided by the protein level of the various NEU1substrates.

If multiple markers are present in a given profile, not all the markersmust show an altered activity as compared to the marker value in acorresponding control or reference profile in order to produce theprognosis and/or diagnosis provided herein. In some instances, thealteration of a single marker may be sufficient for a diagnosis and/orprognosis. In other embodiments, an alteration in 2, 3, 4, 5, 6, 7, 8,9, 10 or more marker values in a given profile as compared to the valuesin corresponding control or reference profile is sufficient for adiagnosis and/or prognosis.

TABLE 1 Summary of various markers employed to establish a specific typeof activity profile. NEU1 Lysosomal Lysosomal Substrate ExocytosisSialylation Sialidase Sialylation NEU1 Level Activity Activity ActivityActivity Activity Marker Profile Profile Profile Profile Profile thelevel of NEU1 + + + + protein the level of direct + + + enzymaticactivity of NEU1 the protein level of one + + + + or more NEU1 At leastone substrates NEU1 substrate other than MUC-1 must be detected theprotein level of + + + + LAMP-1 the protein level of + + + + MUC-1 Onlyin combination with another NEU1 substrate the protein level of + + + +LAMP-1 and MUC-1 the level of any one or + more lysosomal proteins theprotein level of one + or more lysosomal proteases the protein level ofone + or more cathepsins the protein level of + Hexosaminidase beta theprotein level of + mannosidase alpha the activity level of + + + one ormore NEU1 substrates the activity level of + + + LAMP-1 the activitylevel of + + + MUC-1 the activity level of + + + LAMP-1 and MUC-1 theoverall level of + + sialylation in a sample the sialylation levelof + + + + a NEU1 substrate (including levels of a population ofsubstrates and/or the levels of a single substrate) the sialylationlevel of + + + + LAMP-1 the sialylation level of + + + + MUC-1 thesialylation level of + + + + LAMP-1 and MUC-1 the protein leveland + + + the sialylation level of one or more NEU1 substrates the levelof NEU1 + + + protein, the protein level of one or more NEU1 substratesand the sialylation level of one or more NEU1 substrates the level ofNEU1 + + + protein, the level of NEU1 enzymatic activity, the proteinlevel of one or more NEU1 substrates and the sialylation level of one ormore NEU1 substrates

III. Assays for Markers of the Various Activity Profiles

The methods for diagnosis and/or prognosis provided herein are based onanalyzing a sample for lysosomal exocytosis activity, sialylationactivity, lysosomal sialidase activity, NEU1 substrate sialylationactivity and/or NEU1 level activity and comparing it to a referencevalue for lysosomal exocytosis activity, sialylation activity, lysosomalsialidase activity, NEU1 substrate sialylation activity and/or NEU1level activity from a control sample. Measuring the “level” or “amount”of a protein, sialylation, or an activity in a sample means quantifyingthe lysosomal exocytosis activity, sialylation activity, lysosomalsialidase activity, NEU1 substrate sialylation activity or NEU1 levelactivity by determining, for example, the relative or absolute amount ofprotein and/or sialylation of a protein and/or the activity of aprotein. One aspect of the methods provided herein relates to assays fordetecting lysosomal exocytosis activity, sialylation activity, lysosomalsialidase activity, NEU1 substrate sialylation activity and NEU1 levelactivity in the context of a sample. These assays determine the valuesthat make up the lysosomal exocytosis activity profile, sialylationactivity profile, lysosomal sialidase activity profile, NEU1 substratesialylation activity profile or NEU1 level activity profile of a sample.

A “sample” or “subject sample”, as used herein, can comprise any samplein which one desires to determine the lysosomal exocytosis activity,sialylation activity, lysosomal sialidase activity, NEU1 substratesialylation activity and/or NEU1 level activity. By “subject” isintended any animal (i.e. mammals) such as, humans, primates, rodents,agricultural and domesticated animals such as, but not limited to, dogs,cats, cattle, horses, pigs, sheep, and the like, in which one desires todetermine the lysosomal exocytosis activity, sialylation activity and/orlysosomal sialidase activity. The sample may be derived from any cell,tissue, or biological fluid from the animal of interest. The sample maycomprise any clinically relevant tissue, such as, but not limited to,bone marrow, cerebrospinal fluid, tumor biopsy, fine needle aspirate, ora sample of body fluid, such as blood, plasma, serum, lymph, asceticfluid, cystic fluid or urine. The sample used in the methods providedherein will vary based on the assay format, nature of the detectionmethod, and the tissues, cells or extracts which are used as the sample.

A “reference” lysosomal exocytosis activity, sialylation activity,lysosomal sialidase activity, NEU1 substrate sialylation activity and/orNEU1 level activity as used herein is provided in a control sample. A“control” or “control sample” provides a reference point for measuringchanges in lysosomal exocytosis activity, sialylation activity,lysosomal sialidase activity, NEU1 substrate sialylation level activityand/or NEU1 level activity of a subject sample. The control may be apredetermined value based on a group of samples or it may be a singlevalue based on an individual sample. The control may be a sample testedin parallel with the subject sample. A control sample may comprise, forexample: (a) any sample from healthy individual(s); (b) a normal tissuesample taken from a location adjacent to a tumor from the same subject;(b) a tissue sample from healthy individual(s) taken from the sametissue type as a subject tumor; (c) a serum or plasma sample taken fromhealthy individual(s); (d) a cerebrospinal fluid sample taken fromhealthy individual(s); or (e) a urine sample from healthy individual(s).

As used herein a “higher” or “increased” level for a given marker (i.e.any of the various markers provided herein) is meant any significantincrease in the level of the marker in a sample as compared to the levelof the corresponding marker in a control sample. An increased or higherlevel for a given marker can be any statistically significant increasein the level of the marker of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 400%or more as compared to a reference level in a control sample.Alternatively, an increase in the level for a given marker can be anyfold increase of at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold,20-fold or more over the value for the level of the corresponding markerin a control sample. In some embodiments, an increase in the level of agiven marker can result in an increase in a specific activity in thesample (i.e. the lysosomal exocytosis activity, sialylation activity,lysosomal sialidase activity, NEU1 substrate sialylation activity orNEU1 level activity). In other embodiments, an increase in the level ofa given marker can result in a decrease in a specific activity in asample.

As used herein, a “decreased”, “lower” or “reduced” level for a givenmarker (i.e. any of the various markers provided herein) is meant anysignificant decrease in the level of the marker in a sample as comparedto the level of the corresponding marker in a control sample. By loweror reduced level of a marker is meant a statistically significantreduction in the level of a marker in a subject sample of at least 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more as compared to a reference level in a controlsample. Alternatively, a decrease in the level for a given marker can beany fold decrease of at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold,20-fold or more as compared to the level of the corresponding marker ina control sample. In some embodiments, a decrease in the level of agiven marker can result in a decrease in a specific activity in thesample (i.e. the lysosomal exocytosis activity, sialylation activity,lysosomal sialidase activity, NEU1 substrate sialylation activity orNEU1 level activity). In other embodiments, a decrease in the level of agiven marker can result in an increase in a specific activity in thesample.

Table 2 provides non-limiting examples of markers for the variousactivity profiles provided herein and denotes if an increase or adecrease in the marker is reflective of an increase or a decrease in theactivity in a sample (i.e. the lysosomal exocytosis activity,sialylation activity, lysosomal sialidase activity, NEU1 substratesialylation activity or NEU1 level activity).

A. Lysosomal Exocytosis Activity

In one embodiment, the lysosomal exocytosis activity of one or morelysosomal exocytosis activity markers in a sample is provided. As usedherein, “exocytosis” is a process of cellular secretion in whichsubstances contained in vesicles are discharged from the cell by fusionof the vesicular membrane with the outer cell membrane. There are twotypes of exocytosis, constitutive and regulated. Constitutive exocytosisis not regulated by calcium, while regulated exocytosis is dependent oncalcium. Exocytosis involves vesicle recruitment, tethering and dockingof the vesicle to the plasma membrane and fusion of the vesicle membranewith the plasma membrane thereby releasing the contents of the vesicleinto the extracellular space. During exocytosis, the vesicles releasevarious components into the extracellular environment. Some examples ofcomponents of secretory vesicles include, but are not limited to,enzymes, proteases, extracellular matrix components, hormones,neurotransmitters and cytotoxic compounds.

Lysosomal exocytosis is one type of exocytosis. By “lysosomalexocytosis” is meant the process by which lysosomes release theircontents to the extracellular space. Lysosomal exocytosis is a calciumdependent process that involves the recruitment and docking of lysosomesto the plasma membrane, fusion of the lysosomal membrane with the plasmamembrane and the release of lysosomal luminal content into theextracellular environment. Some examples of lysosomal contents include,but are not limited to, enzymes, such as lipases, proteases, nucleasesand amylase, and other proteins related to lysosomal function, such assialidases and proteins involved in lysosomal exocytosis.

In one embodiment, a subject sample has a higher or increased lysosomalexocytosis activity as compared to a control sample. By “higherlysosomal exocytosis activity” or “increased lysosomal exocytosisactivity” is meant a statistically significant alteration in the levelof one or more markers in the lysosomal exocytosis activity profile.Table 2 provides non-limiting examples of markers for the lysosomalexocytosis activity profile and denotes if an increase or a decrease inthe marker is reflective of a higher lysosomal exocytosis activity.

In one embodiment, an increase in lysosomal exocytosis activity isdenoted in a given profile by an alteration in at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the lysosomal exocytosisactivity markers as compared to a control sample. In some cases, analteration in lysosomal exocytosis activity of one marker is sufficientfor a diagnosis and/or prognosis. In other cases an alteration in two ormore lysosomal exocytosis activity markers is sufficient for a diagnosisand/or prognosis.

Assays to measure lysosomal exocytosis activity for an exocytosis markerare provided herein. One measure of lysosomal exocytosis activity is thelevel of a protein in a sample (i.e. NEU1, NEU1 substrates or lysosomalproteins). A variety of assays for detecting protein in a sample areknown in the art and include direct and indirect assays for protein. Anexemplary method for detecting the presence or absence or the quantityof a protein in a sample involves obtaining a sample and contacting thesample with a compound or agent capable of specifically binding anddetecting the protein, such that the presence of the protein is detectedin the sample. Results obtained with a sample from a subject may becompared to results obtained with a biological sample from a controlsubject.

In one embodiment, an agent for detecting a protein is an antibodycapable of specifically binding to that protein. Antibodies can bepolyclonal or monoclonal. The term “labeled”, with regard to theantibody is intended to encompass direct labeling of the antibody bycoupling (i.e. physically linking) a detectable substance to theantibody as well as indirect labeling of the antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody.

The level of a protein in a sample can be quantitatively measured by avariety of assays utilizing antibodies for a specific protein. Theseinclude, for example, immunoassays, radioimmunoassays, enzyme-linkedimmunosorbant assays and two-antibody sandwich assays. Quantitativewestern blotting can also be used to determine the level of protein.Western blots can be quantitated by well-known methods such as scanningdensitometry. In addition, antibodies can be used to detect andquantitate the level of protein in a sample of a tissue by fluorescenceor confocal microscopy by using a fluorescently labeled antibody orsecondary reagent.

In another embodiment, a marker is the level of sialylation of a sample.Assays for measuring the level of sialylation in a sample are providedelsewhere herein, for example, in the section on sialylation activity.

In yet another embodiment, a marker is the level of protein activity ina sample. The protein activity for any protein provided herein can bemeasured by assaying for the activity of the specific protein in asample. For example, if the protein is an enzyme, the activity of theenzyme can be measured in an enzyme activity assay. Various assays areknown in the art for measuring enzymatic activity. For example, NEU1enzyme activity in a sample can be measured by incubating the samplewith a sialylated NEU1 substrate and detecting the amount of free sialicacid present in the sample after incubation. As such, the units ofenzyme activity can be calculated (i.e. the amount of activity permilligram of protein).

B. Sialylation Activity

In one embodiment, the sialylation activity of one or more sialylationactivity markers in a sample is provided. As used herein, a protein orlipid is “sialylated” if a sialic acid is present on the terminalportion of a glycoprotein or glycolipid. By “sialylation” is meant thetransfer of sialic acid to the terminal portions of the sialylatedglycolipids or to the N- or O-linked sugar chains of glycoproteins.Sialylation can be catalyzed by a number of differentsialyltransferases, each with specificity for a particular sugarsubstrate. Non-limiting examples of sialyltransferases known in the artinclude, for example, sialyltransferase, beta-galactosamidealpha-2,6-sialyltransferase, alpha-N-acetylgalactosaminidealpha-2,6-sialyltransferase, beta-galactosidealpha-2,3-sialyltransferase, N-acetyllactosaminidealpha-2,3-sialyltransferase, alpha-N-acetyl-neuraminidealpha-2,8-sialyltransferase and lactosylceramidealpha-2,3-sialyltransferase.

Sialyltransferases can transfer sialic acid to a substrate by variouslinkages. For example, some sialyltransferases add sialic acid with analpha-2,3 linkage to galactose, while other sialyltransferases addsialic acid with an alpha-2,6 linkage to galactose orN-acetylgalactosamine. Another group of sialyltransferases can addsialic acid to other sialic acids by an alpha-2,8 linkage. In oneembodiment, the sialic acid is added with an alpha-2,6 linkage to aglycoprotein. In another embodiment, the sialic acid is added with analpha 2,3 linkage to a glycoprotein.

In one embodiment, a subject sample has a higher or increasedsialylation activity as compared to a control sample. By “highersialylation activity” or “increased sialylation activity” is meant astatistically significant alteration in the level of one or more markersin the sialylation activity profile. Table 2 provides non-limitingexamples of markers for the sialylation activity profile and denotes ifan increase or a decrease in the marker is reflective of a highersialylation activity.

In one embodiment, an increase in sialylation activity is denoted in agiven profile by an alteration in at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or all of the sialylation activity markers ascompared to a control sample. In some cases, an alteration insialylation activity of one marker is sufficient for a diagnosis and/orprognosis. In other cases an alteration in two or more sialylationactivity markers is sufficient for a diagnosis and/or prognosis.

Various assays are known to measure sialylation levels in a sample. Forexample, a sample can be incubated with sambuscus nigra lectin (SNA)that binds preferentially to sialic acid attached to a terminalgalactose in position alpha-2,6. Other assays for sialylation are knownin the art and include the use of Machia amurentis lectin that bindssialic acids attached with an alpha-2,3 linkage.

In one embodiment, sialylation activity can be measured for a populationof proteins to determine global sialylation of proteins in a sample. Forexample, sialylation in this context can be assayed for in a sample bylectin binding assays. The lectin binding assays can be ELISA based orcan be gel based. In another embodiment, the sialylation activity of asingle protein can be measured. In this instance, an ELISA based or gelbased lectin assay can be coupled with a specific antibody to a proteinof interest. The level of sialylation in a sample can be quantitated byusing samples of known different sialylation levels as standards in theassay.

C. Lysosomal Sialidase Activity

In one embodiment, the sialylation activity comprises the level oflysosomal sialidase activity. “Sialidases” are enzymes that remove theterminal sialic acid from glycoproteins by a process calleddesialylation. In mammals, there are at least four types of sialidasesincluding, for example, Neuraminidase 1 (NEU1), NEU2, NEU3 and NEU4which differ in substrate specificity and subcellular localization.NEU1, for example, is localized to the lysosome and cleaves terminalsialic acid residues from substrates such as glycoproteins. As such,NEU1 is an enzyme that contributes to the overall sialylation activityof a sample.

In the lysosome, NEU1 is part of a heterotrimeric complex together withbeta-galactosidase and protective protein/cathepsin A (PPCA). Thepresence of PPCA in the NEU1 complex stabilizes NEU1 in the lysosome.NEU1 has various substrates. As used herein, a “NEU1 substrate” is anyprotein that is desialylated by NEU1. Some non-limiting examples of NEU1substrates include LAMP-1, Cathepsin A, mucins (i.e. MUC1), cathepsin D,cathepsin B and Amyloid Precursor Protein. NEU1 can catalyze thehydrolysis of alpha 2-3 and alpha 2-6 sialyl linkages of terminal sialicacid residues in oligosaccharides, glycoproteins and glycolipids.Desialylation of a glycoprotein, for example, leads to thedestabilization and degradation of the protein. Thus, the sialidase,NEU1, contributes to the turnover of glycoproteins.

In addition to its role as a sialidase, NEU1 has a related effect on theconstitutive process of lysosomal exocytosis. As described elsewhereherein, lysosomal exocytosis involves the recruitment and docking oflysosomes to the plasma membrane, fusion of the lysosomal membrane withthe plasma membrane and the release of lysosomal luminal content intothe extracellular environment. The recruitment and docking step isfacilitated by the lysosomal associated protein-1 (LAMP-1). LAMP-1 is aNEU1 substrate, and thus the stability and turnover rate of LAMP-1 canbe influenced by lysosomal sialidase activity.

In one embodiment, a subject sample has a lower or decreased lysosomalsialidase activity as compared to a control sample. By “lower lysosomalsialidase activity” or “decreased lysosomal sialidase activity” is meanta statistically significant alteration in the level of one or moremarkers in the lysosomal sialidase activity profile. Table 2 providesnon-limiting examples of markers for the lysosomal sialidase activityprofile and denotes if an increase or a decrease in the marker isreflective of a lower lysosomal sialidase activity.

In one embodiment, a decrease in lysosomal sialidase activity is denotedin a given profile by an alteration in at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or all of the lysosomal sialidase activitymarkers as compared to a control sample. In some cases, an alteration inlysosomal sialidase activity of one marker is sufficient for a diagnosisand/or prognosis. In other cases, an alteration in two or more lysosomalsialidase activity markers is sufficient for a diagnosis and/orprognosis.

In one embodiment, lysosomal sialidase activity is measured by the levelof NEU1 protein or enzymatic activity of NEU1. Assays to measure NEU1protein level are well known in the art and include contacting a samplewith an antibody to NEU1. In addition, NEU1 enzymatic activity can bemeasured directly in a sample by assaying for NEU1 enzyme activity of asample in the presence of a sialylated NEU1 substrate. Thus, when NEU1protein and/or enzyme activity levels in a sample are low or absent,NEU1 substrates will not be desialylated or will be desialylated at alower rate resulting in an increase in sialylation of the substrateand/or an increase in the stability of the substrate and thus anincrease in the protein level of the NEU1 substrate in a sample. Forexample, under conditions where NEU1 protein is not present in a sample(i.e. in a NEU1 knockout), LAMP-1 is over-sialylated, accumulates in thelysosome, recruits the lysosome to the plasma membrane and facilitatesdocking of the lysosome to the plasma membrane. In such cases, the lossof NEU1 protein/activity results in an increase in lysosomal exocytosis.

As used herein, an increase in sialylation of any one or more NEU1substrates results in a lower lysosomal sialidase activity in a sample.Further, an increase in the protein level of any one or more of the NEU1substrates provided herein also results in a lower lysosomal sialidaseactivity. As such, these values are markers for lysosomal sialidaseactivity and indicative of low protein and activity levels of NEU1 in asample. Assays to measure for sialylation levels in a sample or thesialylation level of a specific protein in a sample are discussedelsewhere herein. Assays to measure the protein level of any of thevarious lysosomal sialidase activity markers are known in the art andare described in detail elsewhere herein.

In another embodiment, the enzymatic activity level of NEU1 or theactivity level of any of the various NEU1 substrates are markers forlysosomal sialidase activity. Assays to measure the protein activity forvarious proteins is known in the art and described elsewhere herein.

D. NEU1 Substrate Sialylation Activity

In one embodiment, one type of lysosomal sialidase profile is a NEU1substrate sialylation activity profile. Non-limiting examples of thevarious NEU1 substrate sialylation activity markers are summarized inTable 1.

In one embodiment, a subject sample has a higher or increased NEU1substrate sialylation activity as compared to a control sample. By“higher NEU1 substrate sialylation activity” or “increased NEU1substrate sialylation activity” is meant a statistically significantalteration in the level of one or more markers in the NEU1 substratesialylation activity profile. Table 2 provides non-limiting examples ofmarkers for the NEU1 substrate sialylation activity profile and denotesif an increase or a decrease in the marker is reflective of a higherNEU1 substrate sialylation activity.

In one embodiment, an increase in NEU1 substrate sialylation activity isdenoted in a given profile by an alteration in at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the NEU1 substratesialylation activity markers as compared to a control sample. In somecases, an alteration in NEU1 substrate sialylation activity of onemarker is sufficient for a diagnosis and/or prognosis. In other cases analteration in two or more NEU1 substrate sialylation activity markers issufficient for a diagnosis and/or prognosis.

Assays to measure the NEU1 substrate sialylation activity of the variousNEU1 substrate sialylation activity markers are known in the art andinclude measuring the level of sialylation of any of the various NEU1substrates provided herein. Such assays are described elsewhere herein.

E. NEU1 Level Activity

In another embodiment, one type of lysosomal sialidase activity profileis a NEU1 level activity profile. Non-limiting examples of the variousNEU1 level activity markers are summarized in Table 1.

In one embodiment, a subject sample has a higher or increased NEU1 levelactivity as compared to a control sample. By “higher NEU1 levelactivity” or “increased NEU1 level activity” is meant a statisticallysignificant alteration in the level of two or more markers in the NEU1level activity profile. Table 2 provides non-limiting examples ofmarkers for the NEU1 level activity profile and denotes if an increaseor a decrease in the marker is reflective of a higher NEU1 levelactivity.

In one embodiment, an increase in NEU1 level activity is denoted in agiven profile by an alteration in at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or all of the NEU1 level activity markers as comparedto a control sample. In some cases, an alteration in NEU1 level activityof two or more markers is sufficient for a diagnosis and/or prognosis.

Assays to measure NEU1 level activity of the various NEU1 level activitymarkers are known in the art and include, for example, immuno-blottingusing an antibody specific for a NEU1 level activity marker or ELISAassay using an antibody specific for a NEU1 level activity marker. Theseassays are discussed in detail elsewhere herein.

TABLE 2 Summary of alterations in various markers employed to establishan increase or decrease in a specific type of activity. Marker Level ofMarker Compared to Control Increased Lysosomal exocytosis activity thelevel of NEU1 protein Decreased the level of NEU1 enzymatic Decreasedactivity the protein level of one or more Increased NEU1 substrates theprotein level of LAMP-1 Increased the protein level of MUC-1 Increasedthe protein level of any one or Increased more lysosomal proteins theprotein level of one or more Increased lysosomal proteases the activitylevel of one or more Increased NEU1 substrates the activity level ofLAMP-1 Increased the activity level of MUC-1 Increased The overall levelof sialylation Increased in a sample the sialylation level of one orIncreased more NEU1 substrates the sialylation level of LAMP-1 Increasedthe sialylation level of MUC-1 Increased the protein level of one ormore Increased cathepsins the protein level of Increased Hexosaminidasebeta the protein level of mannosidase Increased alpha IncreasedSialylation Activity the level of NEU1 protein Decreased The level ofNEU1 enzymatic Decreased activity the protein level of one or moreIncreased NEU1 substrates the protein level of LAMP-1 Increased theprotein level of MUC-1 Increased the activity level of one or moreIncreased NEU1 substrates the activity level of LAMP-1 Increased theactivity level of MUC-1 Increased the overall level of sialylation inIncreased a sample the sialylation level of one or Increased more NEU1substrates the sialylation level of LAMP-1 Increased the sialylationlevel of MUC-1 Increased Decreased Lysosomal Sialidase Activity thelevel of NEU1 protein Decreased the level of NEU1 enzymatic Decreasedactivity the protein level of one or more Increased NEU1 substrates theprotein level of LAMP-1 Increased the protein level of MUC-1 IncreasedThe activity level of one or more Increased NEU1 substrates the activitylevel of LAMP-1 Increased the activity level of MUC-1 Increased thesialylation level of one or Increased more NEU1 substrates thesialylation level of LAMP-1 Increased the sialylation level of MUC-1Increased Increased NEU1 Substrate Sialylation Activity the sialylationlevel of one or Increased more NEU1 substrates the sialylation level ofLAMP-1 Increased the sialylation level of MUC-1 Increased Increased NEU1Level Activity the protein level of one or more Increased non-MUC-1 NEU1substrates the protein level of MUC-1-only Increased in combination withanother NEU1 substrate the protein level of LAMP-1 Increased the proteinlevel of MUC-1 Increased

IV. Cancer

The various profiles provided herein can be used in methods of prognosisof a chemotherapy regime, diagnosis of cancer and prognosis of cancer ina subject. As provided herein “prognosis” is the likely outcome of apathological condition or disease (i.e. the expected morbidity ormortality, the expected outcome of a therapy, or the risk ofmetastasis). “Diagnosis” refers to determining whether a subject islikely to have a disease or condition.

As mentioned in the previous section, NEU1 is a regulator of lysosomalexocytosis. Moreover, NEU1 is the only known regulator of lysosomalexocytosis. Defects in lysosomal exocytosis have been associated withvarious diseases. For example, NEU1 deficiency results in the lysosomalstorage disease sialidosis.

Under conditions where NEU1 levels or enzyme activity are low, the NEU1substrate, LAMP-1, accumulates in an over-sialylated state in thelysosome. As discussed, LAMP-1 enhances lysosomal exocytosis. As such, alower lysosomal sialidase activity results in an increase in lysosomalexocytosis and release of lysosomal contents into the extracellularspace.

Described herein is the discovery that cancer cells and tumors fromvarious types of cancers have a low lysosomal sialidase activity (i.e.as measured using any of the lysosomal sialidase activity markersprovided herein). See, for example, Example 1 described elsewhereherein. In such cases, the down-regulation of NEU1 leads to aderegulation of lysosomal exocytosis in the cancer cells, thusincreasing lysosomal exocytosis.

Excessive lysosomal exocytosis can have profound effects on cancerdiagnosis, prognosis and chemotherapy as discussed herein. The methodsof determining the prognosis of a lysosomotropic chemotherapeutic agent,and the diagnosis and prognosis of cancer provided herein encompass anytype of cancer in a subject. Non-limiting examples of types of cancerencompassed by the methods herein include, sarcomas, leukemia, lymphoma,breast cancer, colon cancer, rhabdomyosarcoma, Ewing's sarcoma, lungcancer, bladder cancer, pancreatic cancer, ovarian cancer, prostatecancer, brain tumors, acute lymphoblastic leukemia, and bone cancer. Inspecific embodiments, the cancer comprises rhabdomyosarcoma, breastcancer, colon cancer, pancreatic cancer or Ewing's sarcoma.

A. Methods of Prognosis of a Chemotherapy Regime

Provided herein are methods of determining the prognosis for alysosomotropic chemotherapeutic agent regime in a subject with cancer.As used herein, a “lysosomotropic chemotherapeutic agent” is meant anychemotherapeutic agent that accumulates preferentially in the lysosomesof cells. Many commonly used chemotherapeutic agents accumulate in theacidic lysosome due to their weakly basic nature. Some non-limitingexamples of lysosomotropic chemotherapeutic agents include doxorubicin,cisplatin and docetaxel.

In cases where NEU1 is down-regulated in a cancer, this leads to aderegulation of lysosomal exocytosis in the cancer cells, thusincreasing lysosomal exocytosis. As such, chemotherapeutic agents whichaccumulate in the lysosomes are released from the cancer cells into theextracellular space thereby preventing the chemotherapy from having aneffect on the cell. Thus, a low lysosomal sialidase activity ispredictive of chemotherapy resistance to lysosomotropic chemotherapeuticagents. By “resistant” to chemotherapy is meant the ability of a cell ortumor to withstand the effects of a chemotherapeutic agent(s).

The prognosis for a lysosomotropic chemotherapeutic agent regime in asubject with cancer can be determined by obtaining a lysosomal sialidaseactivity profile of a sample from the subject with cancer. In suchcases, an alteration in the lysosomal sialidase activity of any one ormore lysosomal sialidase activity markers as compared to a controlsample, as depicted, for example, in Table 2, results in a lower ordecreased lysosomal sialidase activity for the sample. In the case wherethe lysosomal sialidase activity is lower in the subject sample ascompared to a control sample, it is predicted that the cancer will beresistant to the lysosomotropic chemotherapy.

In one embodiment, the method of determining the prognosis for alysosomotropic chemotherapeutic agent regime in a subject with cancercomprises the steps of: (a) providing a subject lysosomal sialidaseactivity profile from a tumor sample from the subject; (b) providing areference lysosomal sialidase activity profile from a control sample,wherein the subject lysosomal sialidase activity profile and thereference lysosomal sialidase activity profile comprise one or morevalues representing lysosomal sialidase activity; and (c) comparing thesubject and the reference lysosomal sialidase activity profiles tothereby determine the prognosis for a lysosomotropic chemotherapeuticagent regime in the subject, wherein a lower lysosomal sialidaseactivity of the subject as compared to the lysosomal sialidase activityof the reference results in a prediction that the cancer will beresistant to the lysosomotropic chemotherapeutic agent.

In one embodiment, the lysosomal sialidase activity profile comprisesany number and combination of lysosomal sialidase activity values forany of the various lysosomal sialidase activity markers provided herein.Non-limiting examples of the lysosomal sialidase activity profile of asample are provided in Table 1.

In a specific embodiment, the lysosomal sialidase activity comprises thelevel of LAMP-1 protein. In another embodiment, the lysosomal sialidaseactivity comprises the level of MUC-1 protein. In yet anotherembodiment, the lysosomal sialidase activity comprises the level of theNEU1 substrates LAMP-1 and MUC-1. In a further embodiment, the lysosomalsialidase activity comprises the level of LAMP-1 sialylation. In yetanother embodiment, the lysosomal sialidase activity comprises the levelof MUC-1 sialylation. In another specific embodiment, the level oflysosomal sialidase activity comprises the level of LAMP-land MUC-1sialylation. In still further embodiments, the lysosomal sialidaseactivity comprises the level of LAMP-1, the level of MUC-1, the level ofsialylation of LAMP-1 and the level of MUC-1 sialylation.

Knowledge of the lysosomal sialidase activity status of a tumor from asubject will allow the physician to predict the most appropriate therapyfor a subject having a cancer with a low lysosomal sialidase activityprofile. For example, lysosomotropic chemotherapeutic agents would notbe chosen for treating a tumor with low lysosomal sialidase activityprofile since this is predictive that the tumor will be resistant tothese agents. Thus, a treatment regime with chemotherapeutic drugs thatdo not accumulate in the lysosome would be a better treatment option.

B. Methods of Diagnosis and Prognosis of Cancer

The methods herein also provide a method of determining the prognosisand diagnosis for a subject with cancer. Information obtained from thediagnosis and prognosis can be useful in selecting an appropriatetreatment.

As described elsewhere herein, NEU1 is a negative regulator of lysosomalexocytosis and low lysosomal sialidase activity results in an increasein lysosomal exocytosis. Excess lysosomal exocytosis can have profoundeffects on the extracellular environment of a cell. For example, thelysosome contains proteases which breakdown the extracellular matrixresulting in a remodeling of the extracellular environment. Thebreakdown of the extracellular matrix increases the vulnerability oftissue to invasion. As such, a high concentration of proteases in theextracellular matrix surrounding a cancer cell can enhance the invasivepotential and metastasis of a cancer cell.

A cancer that is “invasive” has the ability to spread to the surroundingtissue. “Metastasis”, as used herein, refers to the process by which acancer spreads or transfers from the site of origin to other regions ofthe body. As depicted elsewhere herein, cancers that have low lysosomalsialidase activity have an increased invasive potential. Assays thatmeasure the invasiveness of a cancer are known in the art and an exampleinvasion assay is described in detail in Example 1 provided elsewhereherein.

Invasive cancers are more likely to metastasize and thus have anunfavorable prognosis, whereas non-invasive cancers are less likely tometastasize and therefore have a favorable prognosis. The term“unfavorable prognosis” in regards to tumors or subjects diagnosed withcancer refers to a tumor or subject with a high probability ofmetastasis and/or a high probability of causing death or dying. A“favorable prognosis” in regards to a subject diagnosed with cancerrefers to a tumor or subject with a low probability of metastasis and/ora low probability of causing death or dying.

The lysosomal sialidase activity of a sample, for the purpose ofdiagnosis and prognosis of cancer, can be determined by measuring thevalues for any two or more of the various lysosomal sialidase activitymarkers provided herein. Thus, lower levels of lysosomal sialidaseactivity in a subject sample as compared to a reference lysosomalsialidase activity of a control sample are indicative that a tumor hasincreased invasive potential (i.e. an unfavorable prognosis), whilehigher or normal levels of lysosomal sialidase activity in a subjectsample as compared to a reference lysosomal sialidase activity of acontrol sample are predictive of a less invasive potential (i.e. afavorable prognosis).

In some embodiments the diagnosis and/or prognosis of cancer can bedetermined by measuring the NEU1 substrate sialylation activity or theNEU1 level activity of a sample. These activities can be determined bymeasuring the values for any of the various markers provided in Tables 1and 2. For a NEU1 substrate sialylation activity, a higher level of anyone or more NEU1 substrate sialylation activity markers results in ahigher NEU1 substrate sialylation activity and is indicative of cancerand an unfavorable prognosis. For a NEU1 level activity, a higher levelof any two or more NEU1 substrate activity markers results in a higherNEU1 level activity and is indicative of cancer and an unfavorableprognosis.

In one embodiment, a method of determining the prognosis for a subjectwith a cancer is provided and comprises the steps of: (a) providing asubject lysosomal sialidase activity profile comprising two or morevalues from different lysosomal sialidase activity markers, a NEU1substrate sialylation activity profile or a NEU1 level activity profilefrom a tumor sample from the subject; (b) providing a correspondingreference lysosomal sialidase activity profile comprising two or morevalues from different lysosomal sialidase activity markers, a NEU1substrate sialylation activity profile or a NEU1 level activity profilefrom a control sample, wherein the subject profile and the referenceprofile comprise one or more values representing lysosomal sialidaseactivity, NEU1 substrate sialylation activity or NEU1 level activity;and (c) comparing the subject and the reference lysosomal sialidaseactivity profiles, NEU1 substrate sialylation activity profiles or NEU1level activity profiles to thereby determine the prognosis for thesubject with cancer, wherein a lower lysosomal sialidase activity, ahigher NEU1 substrate sialylation activity or a higher NEU1 levelactivity of the subject as compared to the lysosomal sialidase activity,NEU1 substrate sialylation activity or NEU1 level activity of thereference results in a prediction of an invasive cancer for the subject.

In another embodiment, a method of diagnosing cancer in a subject isprovided, the method comprising: (a) providing a subject profilecomprising a lysosomal sialidase activity profile comprising two or morevalues from different lysosomal sialidase activity markers, a NEU1substrate sialylation activity profile or a NEU1 level activity profilefrom a tumor sample from the subject; (b) providing a correspondingreference profile comprising a lysosomal sialidase activity profilecomprising two or more values from different lysosomal sialidaseactivity markers, a NEU1 substrate sialylation activity profile or aNEU1 level activity profile from a control sample, wherein the subjectprofile and the reference profile comprise one or more valuesrepresenting lysosomal sialidase activity, NEU1 substrate sialylationactivity or NEU1 level activity; and (c) comparing the subject and thereference lysosomal sialidase activity profiles, NEU1 substratesialylation profiles or NEU1 level profiles to thereby determine thediagnosis for the subject, wherein the subject is diagnosed with cancerif the lysosomal sialidase activity of the subject is lower, the NEU1substrate sialylation activity is higher or the NEU1 level activity ishigher than the lysosomal sialidase activity, the NEU1 substratesialidase activity or the NEU1 level activity of the reference.

Knowledge of the level of lysosomal sialidase activity, NEU1 substratesialylation activity or NEU1 level activity in a subject sample allows apractitioner to diagnose a subject as having cancer, predict theaggressiveness of a cancer and thereby select the appropriate therapyfor the subject with cancer.

V. Methods of Diagnosis of Dementia Associated With Alzheimer's Disease

Also provided herein are methods for the diagnosis of dementiaassociated with Alzheimer's disease. Provided herein, is a demonstrationthat the lysosomal exocytosis activity profile of a sample from asubject is predictive of dementia associated with Alzheimer's disease.

As used herein, “dementia associated with Alzheimer's disease” ischaracterized by the standard criteria for dementia as reported in theRecommendations from the National Institute on Aging-Alzheimer'sAssociation workgroups on diagnostic guidelines for Alzheimer's disease.The standard criteria for dementia include (1) cognitive or behavioralsymptoms that interfere with the ability to function at usual activitiesor work, denote a decline from previous functioning and performinglevels and cannot be explained by a major psychiatric disorder ordelirium; (2) detection and diagnosis of cognitive impairment through acombination of history-taking from the patient and a knowledgeableinformant and an objective cognitive assessment; and (3) the cognitiveor behavioral impairment involves two or more of the following: (a)impaired ability to acquire and remember new information; (b) impairedreasoning and handling of complex tasks, poor judgment; (c) impairedvisuospatial abilities; (d) impaired language functions; and (e) changesin personality, behavior or comportment. Dementia associated withAlzheimer's can further have one or more of the followingcharacteristics: (1) meets all criteria for dementia as described above;(2) insidious onset; (3) a history of worsening or cognition by reportor observation; (4) amnestic presentation; and (5) nonamnesticpresentations, such as, language presentation, visuospatial presentationor executive dysfunction. The Alzheimer's disease dementia guidelinesare described in detail in McKhann et al. (2011) Alzheimer's & Dementia7:263-69, herein incorporated by reference in its entirety.

As described elsewhere herein, NEU1 is a negative regulator of lysosomalexocytosis. In such instances when NEU1 protein levels or enzymaticactivity are low, lysosomal exocytosis is enhanced. As shown herein,under conditions where the NEU1 protein level is low, highly sialylatedproteins can be detected in the cerebrospinal fluid (CSF). In suchcases, the composition of the CSF is changed and many of the highlysialylated proteins also have increased levels in the CSF. Theseproteins that are changed in the CSF under conditions of low NEU1protein and activity levels correlate with biomarkers for predictingdementia associated with Alzheimer's disease. For example, amyloidprecursor protein (APP) is shown herein to be a NEU1 substrate andaccumulates in the brain and CSF in a highly sialylated form under lowNEU1 conditions. Lysosomes also comprise proteases that can process APPto form toxic Aβ peptides. Thus, excessive lysosomal exocytosis (i.e.when NEU1 protein or enzymatic activity levels are low) enhances theplaque formation that is characteristic of Alzheimer's disease. See, forexample, Example 3, provided elsewhere herein. Thus, in one embodiment,an increased lysosomal exocytosis activity, as described in detailelsewhere herein, in the CSF can be predictive of dementia associatedwith Alzheimer's disease.

A variety of proteins can have an increased sialylation level and/orhave increased levels in the CSF. In some embodiments, the proteinshaving an increased sialylation level and/or protein level are NEU1substrates. In other embodiments, the proteins having an increasedsialylation level and/or protein level are lysosomal proteins.Non-limiting examples of proteins with increased sialylation and/orprotein level include LAMP-1, MUC-1, Cathepsin B, Cathepsin D,Complement system proteins, Fibrinogen, Hexosaminidase beta, Mannosidasealpha, Transthyretin, beta-2 microglobulin and Amyloid PrecursorProtein. Any one or more of these proteins can be a marker for lysosomalexocytosis activity.

Provided herein is a method of diagnosing dementia associated withAlzheimer's disease in a subject, the method comprising: (a) providing asubject lysosomal exocytosis activity profile of a sample ofcerebrospinal fluid from the subject; (b) providing a referencelysosomal exocytosis activity profile of a control sample ofcerebrospinal fluid, wherein the subject lysosomal exocytosis activityprofile and the corresponding reference lysosomal exocytosis activityprofile comprise one or more values representing lysosomal exocytosisactivity; and (c) comparing the subject and the reference lysosomalexocytosis activity profiles, wherein the subject is diagnosed withdementia associated with Alzheimer's disease if the subject has a higherlysosomal exocytosis activity as compared to the reference lysosomalexocytosis activity.

In one embodiment the lysosomal exocytosis activity profile comprises alysosomal sialidase activity profile. The lysosomal sialidase activityprofile can comprise any combination of any of the various lysosomalsialidase activity markers provided herein. In such cases, a lowlysosomal sialidase activity in a subject sample as compared to areference lysosomal sialidase activity in a control sample results in asubject being diagnosed with dementia associated with Alzheimer'sdisease.

In another embodiment, the lysosomal exocytosis activity profilecomprises a sialylation activity profile. The sialylation activityprofile can comprise any combination of any of the various sialylationactivity markers provided herein. In such cases, a high sialylationactivity in a subject sample as compared to a reference sialylationactivity in a control sample results in a subject being diagnosed withdementia associated with Alzheimer's disease.

For the diagnosis of dementia associated with Alzheimer's disease, thelysosomal exocytosis activity profiles, the lysosomal sialidase activityprofiles or the sialylation activity profiles can comprise any one ormore of the various markers provided herein (i.e. see Tables 1 and 2).

Knowledge of the sialylation activity profile of a subject sample willallow the physician to make a diagnosis of dementia associated withAlzheimer's disease in a subject. Thus, an early diagnosis can be madeand the appropriate treatment options can be considered for the subject.

VI. Methods of Generating a Lysosomal Sialidase Activity Profile and anLysosomal Exocytosis Activity Profile

Methods of generating a lysosomal sialidase activity profile and/or alysosomal exocytosis activity profile for a sample are also provided. Aspresented herein, the lysosomal sialidase activity profile of a samplecan comprise one or more lysosomal sialidase activity markersrepresenting lysosomal sialidase activity (i.e. any of the variousmarkers of lysosomal sialidase activity provided herein, see Table 1).Also herein, the lysosomal exocytosis activity profile of a sample cancomprise one or more lysosomal exocytosis activity markers representinglysosomal exocytosis activity (i.e. any or the various markers oflysosomal exocytosis activity provided herein, see Table 1).

In one embodiment, a method of generating a lysosomal sialidase activityprofile comprises: (a) obtaining a sample from a tumor from a subject;and (b) assaying for the level of LAMP-1 protein or the level of LAMP-1sialylation. In a further embodiment, the method comprises assaying forone or more additional lysosomal sialidase activity markers. In yetanother embodiment of the method, the one or more additional lysosomalsialidase activity markers comprise a NEU1 substrate. Assays formeasuring the various lysosomal sialidase activity markers are providedelsewhere herein.

In another embodiment, a method of generating a lysosomal exocytosisactivity profile from cerebrospinal fluid comprises: (a) obtaining asample of cerebrospinal fluid from a subject; and (b) assaying forlysosomal exocytosis activity. In a specific embodiment, assaying forlysosomal exocytosis activity comprises assaying for the level of LAMP-1protein or the level of LAMP-1 sialylation.

In a non-limiting embodiment, assaying for lysosomal exocytosis activitycomprises assaying for the level of one or more proteins comprisingLAMP-1, MUC-1, amyloid precursor protein, Cathepsin B, Cathepsin D,Fibrinogen, Hexosaminidase beta, Mannosidase alpha, Transthyretin,beta-2 microglobulin or Immunoglobulin heavy chain.

VII. Methods of Treatment

Further provided are methods of treating a subject having a cancer orhaving dementia associated with Alzheimer's disease. By “treating” asubject with cancer or dementia associated with Alzheimer's disease isintended administration of a therapeutically effective amount of NEU1 oran active variant or fragment thereof, administration of atherapeutically effective amount of protective protein/cathepsin A(PPCA) or an active variant or fragment thereof or administration of atherapeutically effective amount of a combination of NEU1 and PPCA to asubject that has cancer or dementia associated with Alzheimer's disease,where the purpose is to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the condition or the symptoms of thecancer or dementia associated with Alzheimer's disease.

Also provided herein are methods of preventing a cancer or dementiaassociated with Alzheimer's disease in a subject. By “preventing” acancer or dementia associated with Alzheimer's disease in a subject isintended administration of a therapeutically effective amount of NEU1 oran active variant or fragment thereof, administration of atherapeutically effective amount of protective protein/cathepsin A(PPCA) or an active variant or fragment thereof or administration of atherapeutically effective amount of a combination of NEU1 and PPCA to asubject, where the purpose is to protect the subject from development ofa cancer or dementia associated with Alzheimer's disease. In someembodiments, a therapeutically effective amount of NEU1 or an activevariant or fragment thereof, protective protein/cathepsin A (PPCA) or anactive variant or fragment thereof or a combination of NEU1 and PPCA isadministered to a subject, such as a human, that is at risk fordeveloping a cancer or dementia associated with Alzheimer's disease.

A “therapeutically effective amount” as used herein refers to thatamount which provides a therapeutic effect for a given condition andadministration regimen. Thus, the phrase “therapeutically effectiveamount” is used herein to mean an amount sufficient to cause animprovement in a clinically significant condition in the host. Inparticular aspects, a “therapeutically effective amount” refers to anamount of NEU1, PPCA, or a combination of NEU1 and PPCA provided hereinthat when administered to a subject brings about a positive therapeuticresponse with respect to the treatment of a subject for a cancer ordementia associated with Alzheimer's disease. A positive therapeuticresponse in regard to treating a cancer includes curing or amelioratingthe symptoms of the disease. In the present context, a deficit in theresponse of the host can be evidenced by continuing or spreading of thecancer. An improvement in a clinically significant condition in the hostincludes a decrease in the size of a tumor, increased necrosis of atumor, clearance of the tumor from the host tissue, reduction oramelioration of metastasis, or a reduction in any symptom associatedwith the cancer. A positive therapeutic response in regard to treating asubject with dementia associated with Alzheimer's disease includescuring or ameliorating the symptoms of the disease. In this context, adeficit in the response of the host can be evidenced by continuing orworsening of the dementia associated with Alzheimer's disease. Animprovement in a clinically significant condition in the host includes adecrease in dementia (i.e. an improvement in memory, judgment,visuospatial abilities, language functions, behavior or any of the othersymptoms of dementia provided elsewhere herein) in the subject.

In particular aspects, a “therapeutically effective amount” refers to anamount of NEU1, PPCA, or a combination of NEU1 and PPCA provided hereinthat when administered to a subject brings about a positive therapeuticresponse with respect to the prevention of a cancer or dementiaassociated with Alzheimer's disease in a subject. A positive therapeuticresponse with respect to preventing a cancer or dementia associated withAlzheimer's disease in a subject, for example, is the prevention ofdevelopment of the disease in a subject.

In one embodiment, a method of treating a subject having a cancercomprises administering to a subject in need thereof a therapeuticallyeffective amount of Neuraminidase 1 (NEU1) having an amino acid sequencewith at least 85% sequence identity to SEQ ID NO: 2 or an active variantor fragment thereof.

In another embodiment, a method of treating a subject with dementiaassociated with Alzheimer's disease comprises administering to a subjectin need thereof a therapeutically effective amount of Neuraminidase 1(NEU1) having an amino acid sequence with at least 85% sequence identityto SEQ ID NO: 2 or an active variant or fragment thereof.

In some embodiments, the methods can further comprise administration ofProtective Protein/Cathepsin A (PPCA) having an amino acid sequence withat least 85% sequence identity to SEQ ID NO: 4.

In other embodiments, the administration of NEU1 and PPCA can beseparate or NEU1 and PPCA can be administered to a subjectsimultaneously. The administration can be by any known method ofadministration as described elsewhere herein. In one embodiment, theadministration of NEU1 and/or PPCA comprises administration of a viralvector comprising a nucleotide sequence having at least 85% sequenceidentity to SEQ ID NO: 1 and/or a nucleotide sequence having at least85% sequence identity to SEQ ID NO: 3.

Active variants and fragments of NEU1 can be used in the methodsprovided herein. Such active variants can comprise at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to SEQ ID NO: 2, wherein the active variants retainbiological activity and hence have sialidase activity. Sialidaseactivity is described in detail elsewhere herein. Active variants ofNEU1 are known in the art. There are over 130 types of neuraminidasesknown from various species ranging from viruses to humans. See, forexample, Monti et al. (2010) Adv. Carbohydr. Chem. Biochem. 64:403-79,herein incorporated by reference in its entirety.

Active variants and fragments of PPCA can be used in the methodsprovided herein. Such active variants can comprise at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to SEQ ID NO:4, wherein the active variants retainbiological activity and hence enhances NEU1 enzymatic activity. Assaysto measure for NEU1 enzymatic activity are described elsewhere herein.Active variants of PPCA are known in the art. See, for example, Galjartet al. (1988) Cell 54(6):755-64, herein incorporated by reference in itsentirety.

VIII. Methods of Administration

The methods of treatment for cancer and dementia associated withAlzheimer's disease provided herein can encompass administration oftreatment via any parenteral route, including, but not limited, tointramuscular, intraperitoneal, intravenous, and the like.

Further, as used herein “pharmaceutically acceptable carriers” are wellknown to those skilled in the art and include, but are not limited to,0.01-0.1 M, or 0.05M phosphate buffer or 0.8% saline. Additionally, suchpharmaceutically acceptable carriers may be aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers such as thosebased on Ringer's dextrose, and the like. Preservatives and otheradditives may also be present, such as, for example, antimicrobials,antioxidants, collating agents, inert gases and the like.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g. fatty acids, waxes, oils). Also comprehendedherein are particulate compositions coated with polymers (e.g.poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions presented herein incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

When administered, compounds are often cleared rapidly from mucosalsurfaces or the circulation and may therefore elicit relativelyshort-lived pharmacological activity. Consequently, frequentadministrations of relatively large doses of bioactive compounds may berequired to sustain therapeutic efficacy. Compounds modified by thecovalent attachment of water-soluble polymers such as polyethyleneglycol, copolymers of polyethylene glycol and polypropylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline are known to exhibit substantiallylonger half-lives in blood following intravenous injection than do thecorresponding unmodified compounds (Abuchowski et al., 1981; Newmark etal., 1982; and Katre et al., 1987). Such modifications may also increasethe compound's solubility in aqueous solution, eliminate aggregation,enhance the physical and chemical stability of the compound, and greatlyreduce the immunogenicity and reactivity of the compound. As a result,the desired in vivo biological activity may be achieved by theadministration of such polymer-compound abducts less frequently or inlower doses than with the unmodified compound.

Dosages.

The sufficient amount may include but is not limited to from about 1μg/kg to about 100 μg/kg, from about 100 μg/kg to about 1 mg/kg, fromabout 1 mg/kg to about 10 mg/kg, about 10 mg/kg to about 100 mg/kg, fromabout 100 mg/kg to about 500 mg/kg or from about 500 mg/kg to about 1000mg/kg. The amount may be 10 mg/kg. The pharmaceutically acceptable formof the composition includes a pharmaceutically acceptable carrier.

The preparation of therapeutic compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as an aerosol of the polypeptide delivered to thenasopharynx or as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

An active component can be formulated into the therapeutic compositionas neutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide) and which are formed with inorganicacids such as, for example, hydrochloric or phosphoric acids, or suchorganic acids as acetic, oxalic, tartaric, mandelic, and the like. Saltsformed from the free carboxyl groups can also be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The component or components of a therapeutic composition provided hereinmay be introduced parenterally, transmucosally, e.g., orally, nasally,pulmonarily, or rectally, or transdermally. Preferably, administrationis parenteral, e.g., via intravenous injection, and also including, butis not limited to, intra-arteriole, intramuscular, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranialadministration. The term “unit dose” when used in reference to atherapeutic composition provided herein refers to physically discreteunits suitable as unitary dosage for humans, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent;i.e., carrier, or vehicle.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer (1990) Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. For example, the protein may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton (1987) CRCCrit. Ref Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507;Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas (1983)J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.(1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howardet al. (1989) J. Neurosurg. 71:105). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, i.e., the brain or a tumor, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlledrelease systems are discussed in the review by Langer (1990) Science249:1527-1533.

A subject in whom administration of an active component as set forthabove is an effective therapeutic regimen for a cancer or dementiaassociated with Alzheimer's disease is preferably a human, but can beany animal. Thus, as can be readily appreciated by one of ordinary skillin the art, the methods and pharmaceutical compositions provided hereinare particularly suited to administration to any animal, particularly amammal, and including, but by no means limited to, domestic animals,such as feline or canine subjects, farm animals, such as but not limitedto bovine, equine, caprine, ovine, and porcine subjects, wild animals(whether in the wild or in a zoological garden), research animals, suchas mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., forveterinary medical use.

In the therapeutic methods and compositions provided herein, atherapeutically effective dosage of the active component is provided. Atherapeutically effective dosage can be determined by the ordinaryskilled medical worker based on patient characteristics (age, weight,sex, condition, complications, other diseases, etc.), as is well knownin the art. Furthermore, as further routine studies are conducted, morespecific information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, is able to ascertain proper dosing. Generally,for intravenous injection or infusion, dosage may be lower than forintraperitoneal, intramuscular, or other route of administration. Thedosing schedule may vary, depending on the circulation half-life, andthe formulation used. The compositions are administered in a mannercompatible with the dosage formulation in the therapeutically effectiveamount. Precise amounts of active ingredient required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual. However, suitable dosages may range from about 0.1 to 20,preferably about 0.5 to about 10, and more preferably one to several,milligrams of active ingredient per kilogram body weight of individualper day and depend on the route of administration. Suitable regimes forinitial administration and booster shots are also variable, but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations of ten nanomolar to ten micromolarin the blood are contemplated.

Administration with Other Compounds.

For treatment of cancer or dementia associated with Alzheimer's disease,one may administer the present active component in conjunction with oneor more pharmaceutical compositions used for treating cancer or dementiaassociated with Alzheimer's disease, including but not limited to (1)chemotherapeutic agents; or (2) other drugs for treating symptoms ofAlzheimer's including domepezil, galantamine, memantine, rivastigmine ortacrine. Administration may be simultaneous (for example, administrationof a mixture of the present active component and a chemotherapeuticagent), or may be in seriatim.

Also contemplated are dry powder formulations comprising at least oneprotein provided herein and another therapeutically effective drug, suchas a chemotherapeutic agent or a drug for treating Alzheimer's disease.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include the component orcomponents (or chemically modified forms thereof) and inert ingredientswhich allow for protection against the stomach environment, and releaseof the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the abovederivatized component or components. The component or components may bechemically modified so that oral delivery of the derivative isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the component molecule itself,where the moiety permits (a) inhibition of proteolysis; and (b) uptakeinto the blood stream from the stomach or intestine. Also desired is theincrease in overall stability of the component or components andincrease in circulation time in the body. Examples of such moietiesinclude: polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline. Abuchowski and Davis (1981)“Soluble Polymer-Enzyme Abducts” In: Enzymes as Drugs, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark,et al. (1982) J. Appl. Biochem. 4:185-189. Other polymers that could beused are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), EUDRAGIT L30D coating, AQUATERICcoating, cellulose acetate phthalate (CAP), EUDRAGIT L coating, EUDRAGITS coating, and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The peptide therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextran and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are FAST-FLO diluent,EMDEX diluent, STA-RX 1500 diluent, EMCOMPRESS diluent and AVICELdiluent.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, EXPLOTAB disintegrant. Sodium starch glycolate, Amberlite,sodium carboxymethylcellulose, ultramylopectin, sodium alginate,gelatin, orange peel, acid carboxymethyl cellulose, natural sponge andbentonite may all be used. Another form of the disintegrants are theinsoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants. Binders may be used to hold the therapeuticagent together to form a hard tablet and include materials from naturalproducts such as acacia, tragacanth, starch and gelatin. Others includemethyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose(CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose(HPMC) could both be used in alcoholic solutions to granulate thetherapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, CARBOWAX4000lubricant, and CARBOWAX 6000 lubricant.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the protein (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

In one embodiment, the method comprises the use of viruses foradministering NEU1 and/or PPCA to a subject. Administration can be bythe use of viruses that express NEU1 and/or PPCA, such as recombinantretroviruses, recombinant adeno-associated viruses, recombinantadenoviruses, and recombinant Herpes simplex viruses (see, for example,Mulligan, Science 260:926 (1993), Rosenberg et al., Science 242:1575(1988), LaSalle et al., Science 259:988 (1993), Wolff et al., Science247:1465 (1990), Breakfield and Deluca, The New Biologist 3:203 (1991)).

A NEU1 and/or PPCA gene can be delivered using recombinant viralvectors, including for example, adenoviral vectors (e.g., Kass-Eisler etal., Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al., Proc.Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum. Gene Ther. 4:403(1993), Vincent et al., Nat. Genet. 5:130 (1993), and Zabner et al.,Cell 75:207 (1993)), adenovirus-associated viral vectors (Flotte et al.,Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such asSemliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat. Nos.4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus vectors(Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali andPaoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, suchas canary pox virus or vaccinia virus (Fisher-Hoch et al., Proc. Nat'lAcad. Sci. USA 86:317 (1989), and Flexner et al., Ann. N.Y. Acad. Sci.569:86 (1989)), and retroviruses (e.g., Baba et al., J. Neurosurg 79:729(1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J.Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993),Vile and Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S.Pat. No. 5,399,346). Within various embodiments, either the viral vectoritself, or a viral particle, which contains the viral vector may beutilized in the methods described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al., J. Virol. 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant herpes simplex virus can beprepared in Vero cells, as described by Brandt et al., J. Gen. Virol.72:2043 (1991), Herold et al., J. Gen. Virol. 75:1211 (1994), Visalliand Brandt, Virology 185:419 (1991), Grau et al., Invest. Ophthalmol.Vis. Sci. 30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992),and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press1997).

When the subject treated with a recombinant virus is a human, then thetherapy is preferably somatic cell gene therapy. That is, the preferredtreatment of a human with a recombinant virus does not entailintroducing into cells a nucleic acid molecule that can form part of ahuman germ line and be passed onto successive generations (i.e., humangerm line gene therapy).

IX. Variants and Fragments

Fragments and variants of the polynucleotides encoding the NEU1 and PPCApolypeptides can be employed in the various methods and compositions ofthe invention. By “fragment” is intended a portion of the polynucleotideand hence the protein encoded thereby or a portion of the polypeptide.Fragments of a polynucleotide may encode protein fragments that retainthe biological activity of the native protein. Thus, fragments of apolynucleotide may range from at least about 20 nucleotides, about 50nucleotides, about 100 nucleotides, about 150, about 200, about 250,about 300, about 350, about 400, about 450, about 500, about 550, about600 and up to the full-length polynucleotide encoding the NEU1 or PPCApolypeptide.

A fragment of a polynucleotide that encodes a biologically activeportion of a NEU1 or PPCA polypeptide will encode at least 15, 25, 30,50, 100, 150, 200, or 250 contiguous amino acids, or up to the totalnumber of amino acids present in a full-length NEU1 or PPCA polypeptide.

A biologically active portion of a NEU1 or PPCA polypeptide can beprepared by isolating a portion of one of the polynucleotides encodingthe portion of the NEU1 or PPCA polypeptide and expressing the encodedportion of the polypeptide (e.g., by recombinant expression in vitro),and assessing the activity of the portion of the NEU1 or PPCApolypeptide. Polynucleotides that encode fragments of a NEU1 or PPCApolypeptide can comprise nucleotide sequence comprising at least 16, 20,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 nucleotides, or up to thenumber of nucleotides present in a full-length NEU1 or PPCA nucleotidesequence disclosed herein.

“Variant” sequences have a high degree of sequence similarity. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the NEU1 or PPCA polypeptides. Variants such as thesecan be identified with the use of well-known molecular biologytechniques, as, for example, polymerase chain reaction (PCR) andhybridization techniques. Variant polynucleotides also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode aNEU1 or PPCA polypeptide. Generally, variants of a particularpolynucleotide will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to that particular polynucleotide as determinedby sequence alignment programs and parameters described elsewhereherein.

Variants of a particular polynucleotide can also be evaluated bycomparison of the percent sequence identity between the polypeptideencoded by a variant polynucleotide and the polypeptide encoded by thereference polynucleotide. Thus, for example, isolated polynucleotidesthat encode a polypeptide with a given percent sequence identity to theNEU1 or PPCA polypeptides set forth herein. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described. Where any given pair ofpolynucleotides is evaluated by comparison of the percent sequenceidentity shared by the two polypeptides they encode, the percentsequence identity between the two encoded polypeptides is at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins arebiologically active, that is they continue to possess the desiredbiological activity of the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Biologically active variants of a NEU1 or PPCA polypeptides will have atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to theamino acid sequence for the native protein as determined by sequencealignment programs and parameters described elsewhere herein. Abiologically active variant of a protein may differ from that protein byas few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as fewas 5, as few as 4, 3, 2, or even 1 amino acid residue.

Proteins may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the NEU1 or PPCA proteins can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferable.

Thus, the polynucleotides used in the invention can include thenaturally occurring sequences, the “native” sequences, as well as mutantforms. Likewise, the proteins used in the methods of the inventionencompass naturally occurring proteins as well as variations andmodified forms thereof. Such variants will continue to possess theability to implement a recombination event. Generally, the mutationsmade in the polynucleotide encoding the variant polypeptide should notplace the sequence out of reading frame, and/or create complementaryregions that could produce secondary mRNA structure. See, EP PatentApplication Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different NEU1 or PPCAcoding sequences can be manipulated to create new NEU1 or PPCApolypeptides possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

X. Sequence Identity

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. It is further to be understood that all base sizesor amino acid sizes, and all molecular weight or molecular mass values,given for nucleic acids or polypeptides are approximate, and areprovided for description.

The subject matter of the present disclosure is further illustrated bythe following non-limiting examples.

TABLE 3 Summary of SEQ ID NOS. SEQ ID NO NA/AA Description 1 NANeuraminidase 1 nucleic acid sequence. 2 AA Neuraminidase 1 amino acidsequence. 3 NA PPCA nucleic acid sequence. 4 AA PPCA amino acidsequence.

Non-limiting examples of methods disclosed herein are as follows:

1. A method of determining the prognosis for a subject with cancer,comprising the steps of

a) providing a subject profile comprising a lysosomal sialidase activityprofile comprising two or more values from different lysosomal sialidaseactivity markers, a NEU1 substrate sialylation activity profile or aNEU1 level activity profile from a tumor sample from said subject;

b) providing a corresponding reference profile comprising a lysosomalsialidase activity profile comprising two or more values from differentlysosomal sialidase activity markers, a NEU1 substrate sialylationactivity profile or a NEU1 level activity profile from a control sample,wherein the subject profile and the reference profile comprise one ormore values representing lysosomal sialidase activity, NEU1 substratesialylation activity or NEU1 level activity; and

c) comparing said subject and said reference lysosomal sialidaseactivity profiles to thereby determine the prognosis for said subjectwith cancer, wherein a lower lysosomal sialidase activity, a higher NEU1substrate sialylation activity or a higher NEU1 level activity of saidsubject as compared to the lysosomal sialidase activity, NEU1 substratesialylation activity or NEU1 level activity of said reference results ina prediction of an invasive cancer for said subject.

2. A method of diagnosing cancer in a subject, the method comprising:

a) providing a subject profile comprising a lysosomal sialidase activityprofile comprising two or more values from different lysosomal sialidaseactivity markers, a NEU1 substrate sialylation activity profile or aNEU1 level activity profile from a tumor sample from said subject;

b) providing a corresponding reference profile comprising a NEU1activity profile comprising two or more values from different lysosomalsialidase activity markers, a NEU1 substrate sialylation activityprofile or a NEU1 level activity profile from a control sample, whereinthe subject profile and the reference profile comprise one or morevalues representing lysosomal sialidase activity, NEU1 substratesialylation activity or NEU1 level activity; and

c) comparing said subject and said reference lysosomal sialidaseactivity profiles to thereby determine the diagnosis for said subject,wherein said subject is diagnosed with cancer if said lysosomalsialidase activity of said subject is lower, the NEU1 substratesialylation activity is higher or the NEU1 level activity is higher thanthe lysosomal sialidase activity, NEU1 substrate sialylation activity orNEU1 level activity of said reference.

3. A method of determining the prognosis for a lysosomotropicchemotherapeutic agent regime in a subject with cancer, comprising thesteps of

a) providing a subject lysosomal sialidase activity profile from a tumorsample from said subject;

b) providing a reference lysosomal sialidase activity profile from acontrol sample, wherein the subject lysosomal sialidase activity profileand the reference lysosomal sialidase activity profile comprise one ormore values representing lysosomal sialidase activity; and

c) comparing said subject and said reference lysosomal sialidaseactivity profiles to thereby determine the prognosis for alysosomotropic chemotherapy agent regime in the subject, wherein a lowerlysosomal sialidase activity of said subject as compared to thelysosomal sialidase activity of said reference results in a predictionthat said cancer will be resistant to said lysosomotropicchemotherapeutic agent.

4. The method of any one of embodiments 1, 2 or 3, wherein the controlsample is from normal tissue adjacent to said tumor from said subject.5. The method of any one of embodiments 1, 2 or 3, wherein the one ormore values representing lysosomal sialidase activity comprise the levelof LAMP-1 protein.6. The method of any one of embodiments 1, 2 or 3, wherein the one ormore values representing lysosomal sialidase activity comprise the levelof LAMP-1 and MUC-1 protein.7. The method of any one of embodiments 1, 2 or 3, wherein the one ormore values representing lysosomal sialidase activity comprise the levelof LAMP-1 sialylation.8. The method of any one of embodiments 1, 2 or 3, wherein the one ormore values representing lysosomal sialidase activity comprise the levelof MUC-1 sialylation.9. The method of any one of embodiments 1, 2 or 3, wherein the one ormore values representing lysosomal sialidase activity comprise the levelof LAMP-1 and MUC-1 sialylation.10. The method of any one of embodiments 1, 2 or 3, wherein the one ormore values representing lysosomal sialidase activity comprise the levelof LAMP-1, the level of MUC-1 protein, the level of LAMP-1 sialylationand the level of MUC-1 sialylation.11. The method of embodiment 1, wherein the one or more valuesrepresenting lysosomal sialidase activity comprise the level of MUC-1protein.12. The method of any one of embodiments 1-11, wherein the cancercomprises rhabdomyosarcoma, breast cancer, colon cancer, pancreaticcancer, acute lymphoblastic leukemia or Ewing's sarcoma.13. A method of diagnosing dementia associated with Alzheimer's diseasein a subject, the method comprising:

a) providing a subject lysosomal exocytosis activity profile of a sampleof cerebrospinal fluid from said subject;

b) providing a reference lysosomal exocytosis activity profile of acontrol sample of cerebrospinal fluid, wherein the subject lysosomalexocytosis activity profile and the corresponding reference lysosomalexocytosis activity profile comprise one or more values representinglysosomal exocytosis activity; and,

c) comparing said subject and said reference lysosomal exocytosisactivity profiles, wherein said subject is diagnosed with dementiaassociated with Alzheimer's disease if the subject has a higherlysosomal exocytosis activity as compared to the reference lysosomalexocytosis activity.

14. The method of embodiment 13, wherein said subject lysosomalexocytosis activity profile and said reference lysosomal exocytosisactivity profile comprise

(a) a lysosomal sialidase activity profile, wherein the subjectlysosomal sialidase activity profile and the corresponding referencelysosomal sialidase activity profile comprise one or more valuesrepresenting lysosomal sialidase activity; or

(b) a sialylation activity profile, wherein the subject sialylationactivity profile and the corresponding reference sialylation activityprofile comprise one or more values representing sialylation activity.

15. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of LAMP-1sialylation.16. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of MUC-1sialylation.17. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of LAMP-1and MUC-1 sialylation.18. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of amyloidprecursor protein sialylation.19. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprises the level ofsialylation of one or more proteins comprising LAMP-1, MUC-1, amyloidprecursor protein, Cathepsin B, Cathepsin D, Fibrinogen, Transthyretin,beta-2 microglobulin or Immunoglobulin heavy chain.20. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of LAMP-1protein.21. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of MUC-1protein.22. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of amyloidprecursor protein.23. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of LAMP-1and MUC-1 protein.24. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprise the level of LAMP-1protein, the level of MUC-1 protein, the level of LAMP-1 sialylation andthe level of MUC-1 sialylation.25. The method of embodiment 13, wherein the one or more valuesrepresenting lysosomal exocytosis activity comprises the level ofprotein of one or more proteins comprising LAMP-1, MUC-1, amyloidprecursor protein, Cathepsin B, Cathepsin D, Fibrinogen, Hexosaminidasebeta, Mannosidase alpha, Transthyretin, beta-2 microglobulin orImmunoglobulin heavy chain.26. A method of treating a subject having a cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of Neuraminidase 1 (NEU1) polypeptide having an amino acidsequence with at least 85% sequence identity to SEQ ID NO: 2, whereinsaid polypeptide has sialidase activity.27. A method of treating a subject with dementia associated withAlzheimer's disease comprising administering to a subject in needthereof a therapeutically effective amount of Neuraminidase 1 (NEU1)polypeptide having an amino acid sequence with at least 85% sequenceidentity to SEQ ID NO: 2, wherein said polypeptide has sialidaseactivity.28. The method of any one of embodiments 26 or 27, further comprisingthe administration of Protective Protein/Cathepsin A (PPCA) polypeptidehaving an amino acid sequence with at least 85% sequence identity to SEQID NO: 4, wherein said PPCA polypeptide enhances NEU1 enzymaticactivity.29. The method of embodiment 28, wherein the NEU1 polypeptide and PPCApolypeptide are administered separately or simultaneously.30. The method of embodiment 29, wherein administration of the NEU1polypeptide comprises administration of a viral vector comprising anucleotide sequence having at least 85% sequence identity to SEQ ID NO:1.31. A method of generating a lysosomal sialidase activity profilecomprising:

(a) obtaining a sample from a tumor from a subject; and,

(b) assaying for the level of LAMP-1 protein or the level of LAMP-1sialylation.

32. The method of embodiment 31, comprising assaying for one or moreadditional lysosomal sialidase activity markers.33. The method of embodiment 32, wherein the one or more additionallysosomal sialidase activity markers comprise a NEU1 substrate.34. A method of generating a lysosomal exocytosis activity profile fromcerebrospinal fluid comprising:

(a) obtaining a sample of cerebrospinal fluid from a subject; and,

(b) assaying for lysosomal exocytosis activity.

35. The method of embodiment 34, wherein assaying for lysosomalexocytosis activity comprises assaying for the level of LAMP-1 proteinor the level of LAMP-1 sialylation.36. The method of embodiment 34, wherein assaying for lysosomalexocytosis activity comprises assaying for the level of one or moreproteins comprising LAMP-1, MUC-1, amyloid precursor protein, CathepsinB, Cathepsin D, Fibrinogen, Hexosaminidase beta, Mannosidase alpha,Transthyretin, beta-2 microglobulin or Immunoglobulin heavy chain.37. The method of any one of embodiments 31-33, comprising assembling alysosomal sialidase activity profile in view of the activity valuesobtained.38. The method of any one of embodiments 34-36, comprising assembling alysosomal exocytosis activity profile in view of the activity valuesobtained.39. The method of any one of embodiments 1-38, wherein the subject is ahuman.

The subject matter of the present disclosure is further illustrated bythe following non-limiting examples.

EXPERIMENTAL Overview

We have discovered a novel association between lysosomal sialidaseNEU1-regulated lysosomal exocytosis and two pathological states: (1)cancer and (2) Alzheimer's disease. The loss of NEU1 results inaccumulation of its substrate LAMP-1, which, in turn, facilitates theexocytosis of lysosomal contents. The physiological consequences of thisdepend on the affected tissue. For instance, release of active proteasesinto the extracellular environment may cause remodeling of tissuesurrounding a tumor. In the brain, this release may result in processingof amyloidogenic proteins and formation of plaques. In addition,xenobiotics, which accumulate in the lysosome may undergo efflux throughthis mechanism, altering drug metabolism. This application is ofparticular importance as a possible predictor of chemotherapy resistancein cancer cells and as prognostic marker of dementia related toAlzheimer's disease.

We have identified two read-outs for the loss of NEU1 which may be usedto visualize NEU1 deficiency. These are substrates of NEU1, mucins andthe aforementioned LAMP-1. We suggest that this is actually a possibleproxy marker for NEU1 deficiency/downregulation and, therefore,increased lysosomal exocytosis. Thus, this marker, when combined withother NEU1 substrates, could be indicative of deregulated lysosomalexocytosis of cancer, which predicts both invasiveness and chemotherapyresistance.

Measuring NEU1 expression or catalytic activity in a cancer biopsy mayhave two somewhat related prognostic applications: 1) determine thestate of the cancer: higher NEU1 activity=less aggressive/betterprognosis; lower NEU1 activity=more aggressive/poorer prognosis; and 2)predict responsiveness to chemotherapy: higher NEU1 activity=lesslysosomal exocytosis/less drug efflux extracellularly/more responsive;lower NEU1 activity=increased lysosomal exocytosis/more drug efflux/lessresponsive.

Alternatively, this information can be gleaned via a panel of NEU1substrates. Accumulation of substrate glycoproteins in theiroversialylated state as well as of active lysosomal enzymes indicate aglobal change in processing rather than a discrete upregulation inexpression, which is currently assumed. This global change could thenpredict outcomes and could be used as fingerprint of invasive-low NEU1tumors

This discovery also has therapeutic application. By restoring thenegative regulation of lysosomal exocytosis, cancer cells can becomemore treatable with chemotherapeutic drugs and less aggressive at thesame time. This may be possible by administering NEU1 itself or byadministering its stabilizing partner, protective protein/cathepsin A(PPCA), or by other means, i.e. LAMP1 downregulation.

Likewise, the downstream effects of lysosomal exocytosis can be used asa molecular fingerprint for Alzheimer's pathology. For instance, highlevels of active lysosomal enzymes and other potential substrates ofNEU1 in cerebral spinal fluid allow diagnosis of dementia related tolate onset Alzheimer's disease in patients, filling a gap in patientcare which currently exists.

This is a new approach for distinguishing more aggressive from lessaggressive cancers that can help guide therapeutic decision-making. Thisinvention also can lead to new cancer treatments and neurodegenerationtherapies that may complement existing techniques or provide completelynovel approaches.

Example 1: NEU1 Deficiency in Cancer Development, Progression, andChemotherapy Resistance

Deficiency of the lysosomal sialidase NEU1 results in the lysosomalstorage disease sialidosis. Type I sialidosis is a catastrophicpediatric disease while Type II, or adult onset sialidosis, is arelatively mild condition caused by gene mutations which preserveresidual activity of NEU1. Our own research into NEU1 deficiency,performed in the mouse model of sialidosis, has revealed a novelfunction of NEU1 as an inhibitor of lysosomal exocytosis. In the absenceof NEU1, its substrate LAMP-1 accumulates, increasing the number oflysosomes docked at the PM and ready to engage in lysosomal exocytosis.As a result, lysosomal contents, including active proteases such ascathepsins, are aberrantly released extracellularly, most likelyimpacting the extracellular matrix structure and composition. Wehypothesized that this phenotype could be advantageous for cancer cells,which extensively modify their extracellular matrix. We have thereforeexamined the expression of NEU1 in a variety of cancer cell lines fromfour cancer types: breast carcinoma, colon carcinoma, Ewing's sarcoma,and alveolar rhabdomyosarcoma. For each type of cancer examined, lowerlevels of NEU1 activity correlated with increased expression ofover-sialylated LAMP-1. Here we report on a correlation between alow-NEU1, highly exocytic phenotype and the invasive capacity of cells.In some cases, the invasiveness of tested cell lines was known. Forinstance, the syngeneic system of SW480 and SW620 colon cancer lines iscomposed of cells derived from a primary or metastatic tumor,respectively, from the same patient. In other cases, such as for Ewingssarcoma and rhabdomyosarcoma, invasive potential of the tested celllines was determined in our hands using an ex vivo model of peritonealinvasion. These studies establish a new paradigm for understanding thespread of cancer: invasive potential is enhanced by degradation ofextracellular matrix via lysosomal exocytosis of active proteases.

Lysosomal exocytosis is part of constitutive cellular physiology whichhas particular importance for cancer cells. Translocation of lysosomalcontents to the extracellular matrix (ECM) results in ECM remodeling andincreased vulnerability of healthy tissue to invasion. In addition, manycommonly used chemotherapeutics accumulate in the acidic lysosome due totheir weakly basic nature. Lysosomal exocytosis therefore constitutes amethod of xenobiotic efflux, relieving cancer cells of toxic burden. Wehave characterized the lysosomal enzyme Neuraminidase 1 as a negativeregulator of lysosome exocytosis and here demonstrate the downregulationof NEU1 in several cancer types as well as the advantageousphysiological consequences for cancer cells associated with the loss ofNEU1.

In addition to its canonical role as a sialidase, NEU1 has a related andprofound effect on the constitutive process of lysosomal exocytosis.This functionality is mediated by the NEU1 substrate LysosomalAssociated Membrane Protein 1 (LAMP1) which is left hyper-glycosylatedin the absence of NEU1. This hyper-glycosylated state of LAMP1 appearsto facilitate lysosomal docking at the plasma membrane (PM) andsubsequent exocytosis causing a range of significant physiologicalchanges to both the affected cell and its environment. Here we presentdata to demonstrate that loss of NEU1 and exacerbation of LEX result intwo major physiological shifts in cancer cells: enhanced invasivepotential and increased resistance to chemotherapy.

To first establish a general role for NEU1 in human cancer, we probedmultiple tumor arrays for both NEU1 and two of its natural substrates,LAMP-1 and mucins. We found that downregulation of NEU1 in tumorscompared to healthy tissue was occurred across cancer types and thatusing either LAMP-1 or mucin staining functioned as proxy markers forthis change (data not shown). The finding is significant in part becausethe mucin MUC-1 has long been used as a trusted cancer marker and thisresearch suggests that it may be downstream of another change with manyother predictable, functional consequences.

In order to test functional consequences of changes to NEU1 levels incancer, we evaluated several cell line systems (data not shown). Ineach, we assessed the NEU1 activity in lysates and the correspondingabundance of over-decorated LAMP-1. For each set evaluated, the relativeinvasive potential was determined either from literature or frommatrigel invasion assays. The more invasive cells consistentlydemonstrated reduced NEU1 activity and increased LAMP1 levels comparedto less invasive cells of the same cancer type (data not shown). Wechose the RH41 and RH30 cell lines for further study because thesealveolar rhabdomyosarcoma lines arise from skeletal muscle, along-standing interest of our lab. The relatively high levels of NEU1 inRH41 cells compared to RH30 cells were further confirmed by archivedmicroarray data, real time PCR, Western blot, and immunofluorescence. Inaddition to reduced LAMP-1 in RH41 cells, the high level of NEU1correlates with a reduction of lysosomal exocytosis as measured by mediaactivity assay and TIRF imaging (data not shown). To test the importanceof NEU1 expression on these physiological markers, we generated stableclones of each line, with upregulation of NEU1 in RH30 cells anddownregulation in RH41 cells, along with empty vector controls for each.Once the expression level of each line was recapitulated in the other,we tested for exocytosis changes via LAMP-1 and TIRF imaging (as well asactivity assays). As predicted, we robustly demonstrated that NEU1 is anegative regulator of lysosomal exocytosis in the cancer cell context,marked by an accumulation of LAMP-1 (data not shown).

There are several immediate implications for identifying a regulator oflysosomal exocytosis in cancer cells. This process has been shown tocontribute to both invasive potential and chemotherapy resistance,although a specific target for altering this process has not beenproposed until NEU1. Lysosomal exocytosis is the most likely method forthe efflux of active lysosomal enzymes into the extracellular matrix,and the presence of enzymes such as cathepsin B in the ECM correlateswith metastasis across cancer types. Active proteases participate in ECMremodeling and inhibit the microenvironment's ability to contain tumorspread. Therefore, we tested the stable lines for their ability toinvade a matrigel substrate, primarily composed of laminin and collagenIV, both susceptible to digestion by lysosomal enzymes such ascathepsins.

The stable clone lines for RH41 and RH30 were each plated onto matrigelplugs for two days. The plugs were then fixed, embedded, sectioned, andstained with H&E to visualize the ingress of cells into the substrate(data not shown). Regardless of parental line, those clones with lowNEU1 successfully invaded the matrigel after two days whereas thosecells with high NEU1 were excluded from the gel. This experimentestablished that the NEU1 status of cancer cells can determine thepotential of cells to invade ECM.

However, we further wished to examine the longer term impact oflysosomal exocytosis on non-malignant tissue. We hypothesized that at atumor border, excessive lysosomal exocytosis from the cancer wouldcondition surrounding tissue for invasion, making healthy tissue morevulnerable to a metastatic event. We therefore tested the ability of theparental RH30 and RH41 cells to invade in an ex-vivo setting usingperitoneum harvested from wild-type or Neu1-knockout mice. Tissuescollected from Neu1 knockout mice have undergone constitutive excessivelysosomal exocytosis and can therefore represent the healthy tissue atthe border of cancer undergoing excessive lysosomal exocytosis due toNEU1 downregulation. In fact, a the more invasive RH30 cells were ableto cross into the peritoneum of wild type animals while the RH41 cellswere largely excluded, recapitulating the results from the matrigelassay. Importantly, the less invasive RH41 cells were able to invade theknock out peritoneum as successfully as RH30 cells invaded the wildtype. This result suggests that the damage done by long term lysosomalexocytosis sensitizes tissue to invasion, independent of theaggressiveness of the cancer. Furthermore, RH30 cells placed on knockoutperitoneum resulted in the most aggressive rates on invasion. Therefore,we conclude that lysosomal exocytosis does significant damage to ECM,regardless of the source of the exocytosis. In the case of cancer cells,those with higher rates of exocytosis more successfully invade astandard substrate.

The second prediction for functional changes downstream of NEU1 alsoproved to be relevant to rhabdomyosarcoma. The RH30 line has a baselineresistance to doxorubicin which can be weakened by the addition of NEU1.Conversely, RH41 cells are highly susceptible to doxorubicin and canacquire resistance upon upregulation of NEU1. This result pointsspecifically to the efflux of the drug through the lysosome for a numberof reasons. First, doxorubicin, like many chemotherapeutics, is a weakbase which accumulates in the acid lysosomal compartment. Secondly,neither of these cells lines expresses p-glycoprotein, the traditionallystudied method of drug efflux. Instead, we were able to image thetrafficking of doxorubicin over a 12 hour period and observed that (1)lysosomes condense around the nucleus in RH41 cells prior to collapse ofthe cell (2) the lysosomal enzyme cathepsin B translocates to thenucleus prior to apoptosis, (3) the doxorubicin load in these cells isvirtually entirely held within the nucleus and (4) resistant cellsmaintain a mobile fraction of doxorubicin in lysosomes (data not shown).

Alteration of NEU1 status reversed these trends; although it did notcompletely sensitize the RH30 cells to apoptosis, PARD cleavage could beobserved at previously harmless doses of doxorubicin (data not shown).We decided to chemically inhibit lysosomal exocytosis to see if completeinhibition could fully eliminate doxorubicin resistance. To do this weused verapamil, a calcium channel blocker which has previously beenconsidered an inhibitor of p-glycoprotein. However, recent work hasshown that this drug can sensitize cells to drug regardless ofp-glycoprotein status, suggesting that another mechanism may be the realtarget of the drug. Because lysosomal exocytosis is dependent on Ca++influx, chelation of calcium would chemically halt the process andprovide a testable change. Rh30 cells co-incubated with verapamil anddoxorubicin are fully sensitized to the drug, recapitulating thephenotype of RH41 cells (data not shown). Doxorubicin can be visualizedalmost exclusively in the nucleus of the verapamil-sensitized RH30cells, and the elimination of the mobile fraction of the drug isdemonstrated (data not shown).

In conclusion, we have presented a model whereby lysosomal exocytosis,as regulated by NEU1, is a critical determinant in cancer cell phenotype(see FIG. 1). The loss of NEU1 results in accumulation of its substratesand alterations to baseline physiology. Two consequences oftranslocation of lysosomal contents to the extracellular space aredegradation of the ECM and efflux of lysosomally-accumulatingchemotherapy drugs. Thus, downregulation of NEU1 may be an importantpredictor for resistance to a class of chemotherapy drugs, including thecommonly used doxorubicin, cisplatin, and docetaxel, all of which knownto localize at least in part to the lysosome. In addition, NEU1substrates may represent a rationally designed panel of cancer markers,adding sensitivity to the growing field.

In addition, when taken together, these data predict that geneticdeficiency of NEU would render people more vulnerable to acquiringcancers and that those cancers would tend toward aggressiveness.

Example 2: The Role of NEU1 in Chemotherapy Resistance Background

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy inchildren. For children diagnosed with metastatic disease, 3-yearsurvival rates are only about 30%. Systemic chemotherapy is currentlythe predominant treatment for these patients—and several combinationprotocols are being used on site here at St. Jude—but drug resistanceoften blunts response. We have recently developed a novel hypothesis forhow drug resistance arises and here propose work to clarify themechanism. In brief, chemotherapy drugs often accumulate in lysosomes,which are multi-functional acidic organelles. Lysosomes can then betransported to the cell periphery, fuse their membrane with the plasmamembrane and release their contents into the extracellular space. Thisprocess, called lysosomal exocytosis (LEX), could effectively limitintracellular exposure to drug. Recent work in our lab has identifiedthe lysosomal sialidase NEU1 as a negative regulator of LEX. WithoutNEU1, its substrate LAMP1 (for Lysosome Associated Membrane Protein)accumulates in lysosomes and aids in their translocation to the plasmamembrane. Our preliminary work on RMS cell lines has shown that stableknockdown of NEU1 results in high levels of LAMP1, more LEX, and moredrug resistance. Conversely, upregulation of NEU1 results in less LAMP1,less LEX, and reduced drug resistance. Thus, NEU1 downregulation may beadvantageous for cancer cells and we have observed such a loss inpediatric RMS tumor samples. Simply administering NEU1 to patients maynot be feasible. The NEU1 protein requires complexing with itschaperone, Protective Protein Cathepsin A (PPCA), which may prove to bea rate-limiting step. However, enhancing PPCA is known to significantlyboost NEU1 residual activity and this may prove to be a more tractableentry point into clinical control of LEX.

Hypotheses and Specific Aims:

Downregulation of lysosomal exocytosis will enhance RMS response tochemotherapy. We intend to leverage our understanding of LEX to identifyopportunities for treatment enhancement in the following two aims. (1)Determine if PPCA upregulation enhances outcome via increasing NEU1activity. Our lab has developed an AAV-based delivery method for PPCA,which is entering clinical trials as an enzyme replacement approach forchildren with galactosialidosis. This work suggests that the vector maybe relevant to cancer treatment, as well. (2) Determine if targetingLAMP1 results in improved outcome. A small portion of LAMP1 is availableto bind to trafficking machinery and facilitate peripheral movement oflysosomes. We will use intracellular delivery of antibody against thissequence to competitively bind and limit movement of the organelles.Success with this methodology will validate the LAMP1 site as a possibletarget for small molecule development.

Design:

These experiments, as proof-of-principle in vitro work, will occur inwell-characterized alveolar RMS cell lines. For Specific Aim 1, dosecurves of AAV-PPCA and a panel of chemotherapy drugs (doxorubicin,cisplatin, vincristine) will be tested for induction of apoptosis. Thesame panel will be used in Specific Aim 2, along with two concentrationsof LAMP1 antibody according to established protocols for intracellulardelivery. For each intervention, LEX will be measured by assaying levelsof lysosomal enzymes in culture media.

Potential Impact:

This work will establish LEX as a determinant of chemotherapy outcome.The suite of proteins we examine, PPCA, NEU1, and LAMP1, may then all beused as markers to characterize a given patient's tumor for LEXcapacity. Secondly, the research is expected to indicate possibletargeted methods for inhibiting LEX. Not only could this have an impacton cancer treatment generally, it directly addresses the main hurdle intreating pediatric alveolar RMS, particularly once metastasized.

Background:

Chemotherapy resistance is the key problem facing children withmetastatic rhabdomyosarcoma. Cancer cells can evade chemotherapy by“pumping” the drug out. For instance, drugs can accumulate in organellescalled lysosomes, which can then move to the cell surface, fuse with thecell membrane and release their contents to the outside in a processknown as lysosomal exocytosis. Here we examine the main players in thisprocess and study how to inhibit it so that chemotherapeutic drugsremain inside targeted cells and provoke their demise. First, PPCA is alysosomal protein that guides the enzyme NEU1 into the lysosome andenhances its activity. NEU1 then helps to degrade LAMP1, one of itstarget substrates. This latter step is important to avoid LAMP1accumulation in lysosomes, which in turn causes excessive exocytosis.Here we propose testing methods to affect the upstream (PPCA) anddownstream (LAMP1) players in order to inhibit exocytosis and therebyallow cancer cells to retain the tested drugs.

Hypotheses and Specific Aims:

Lysosomal exocytosis of drugs decreases effectiveness of chemotherapybut this can be reversed by upregulating PPCA or downregulating LAMP1.Specific Aim1 will test upregulation of PPCA using a virus deliverymethod. Specific Aim 2 will test inhibition of LAMP1 through use of anantibody against its binding site so that it cannot facilitate lysosomalmovement.

Potential Impact:

This research is expected to establish lysosomal exocytosis as a majordeterminant of chemotherapy responsiveness. Understanding this mechanismwill allow clinicians to predict tumor exocytic capacity and tailor drugcombinations/doses accordingly. In addition, we hope to establishspecific, potentially druggable targets to inhibit lysosomal exocytosisand enhance patient response.

Example 3: Early Stage Alzheimer's Disease-Phenotype Linked toDeficiency of the Lysosomal Sialidase Neu1

Lysosomal sialidase NEU1 catalyses the hydrolysis ofsialo-glycoconjugates by removing their terminal sialic acid residues.In humans, primary or secondary deficiency of this enzyme leads to twoclinically similar neurodegenerative lysosomal storage disorders:sialidosis and galactosialidosis. Mice deficient in Neu1 recapitulatethe early-onset severe form of sialidosis. We have discovered that lossof Neu1 activity exacerbates the process of lysosomal exocytosis invarious cell types by influencing the sialic acid content of Lamp-1.This increases the ability of a pool of lysosomes to dock at the PM andengage in lysosomal exocytosis. In this study we have investigatedwhether excessive lysosomal exocytosis underlies some of theneurological aspects seen in the brain of Neu1^(−/−) mice.Histopathological examination of the brain of these mice revealed aprogressive and time dependent deposition of inclusions/depositscontaining APP/Aβ peptide, particularly in the CA3 region of thehippocampus and the adjacent fimbria. The affected regions coincide withsites of high Neu1 expression in wild-type brain. This abnormality wasparalleled by abnormal expression of oversialylated Lamp-1 and activatedproteases, both features linked to excessive lysosomal exocytosis. Thesefindings represent an example of a spontaneously occurring AD-likephenotype in a mouse model of a neurodegenerative disease and couldcontribute to the understanding of some of the pathological mechanismsof Alzheimer's disease.

Lysosomal storage diseases (LSDs) comprise a group of more than 50genetic disorders of lysosomal function, mostly caused by defects in oneof the glycan-cleaving lysosomal hydrolases. Enzyme deficiency usuallyleads to impaired substrates' degradation and to their accumulation incells of multiple systemic organs and the nervous system. Here wepresent evidence that mice lacking the lysosomal sialidase Neu1 besidesrecapitulating the neurodegenerative LSD sialidosis, developpathological and molecular changes in the brain, which are reminiscentof early-stage Alzheimer's disease (AD). Consequent to Neu1loss-of-function the combined occurrence of excessive lysosomalexocytosis of neural cells and accumulation of oversialylated Neu1substrates, including the amyloid precursor protein (APP), underliesthis pathogenic cascade. These findings uncover previously unknownmolecular mechanisms that could contribute and/or predispose to AD.

The fundamental role of the mammalian lysosomal sialidase NEU1 is toinitiate the hydrolysis of sialoglyconjugates by removing their terminalsialic acids. This activity is crucial to cell homeostasis becausegenetic defects that alter NEU1 activity disrupt lysosomal metabolismand result in the LSD sialidosis. We have recently identified Neu1 asthe only negative regulator of the physiological process known aslysosomal exocytosis (LEX). The latter is a Ca²⁺-dependent, regulatedmechanism that involves recruitment/docking of lysosomes to the plasmamembrane (PM), a step which is facilitated by the lysosomal associatedprotein-1 (LAMP-1) and is followed by the fusion of the lysosomalmembrane with the PM, and the release of lysosomal luminal content intothe extracellular space. We have shown that loss of Neu1 in mouse BMmacrophages increases the pool of lysosomes, decorated by oversialylatedLAMP-1 on their LM, which are poised to become exocytic. The ensuingexacerbation of this process leads to disease.

Here we tested if Neu1-dependent increase in LEX is the underlyingmolecular mechanism responsible for neurodegeneration in the mouse modelof sialidosis (Neu1^(−/−)). We found that Neu1 was present throughoutthe brain parenchyma but was predominantly expressed in two regions ofthe wild-type brain: the hippocampus and the choroid plexus (CP) (datanot shown). The CP is the exocytic structure of the brain, producing andsecreting the cerebrospinal fluid (CSF), and functions as a barrierinterface between the blood and the CSF. In the KO mice the CP underwentovert morphologic changes associated with extensive vacuolization andexpansion of the lysosomal system (data not shown). This phenotype wasaccompanied by increased expression of a long-lived oversialylatedLamp-1 (data not shown), a target substrate of Neu1. We have shown thisfeature in other cells and tissues of the Neu1^(−/−) mice anddemonstrated it can be used as read-out of excessive LEX. This wasconfirmed by measuring the activity of the lysosomal enzymesα-mannosidase and β-hexosaminidase that were both increased in the KOCSF (data not shown).

We reasoned that excessive exocytosis of lysosomal content into the CSFwould dramatically alter its composition. We investigated thispossibility by comparing the total protein content of the Neu1^(−/−) andNeu1^(+/+) CSF samples using high throughput proteomic analysis. Wefound many lysosomal enzymes, including cathepsin D and cathepsin B, aswell as other proteins to be present in abnormal amounts in the KO CSF(FIG. 2). Increased levels of several of these proteins were alsoobserved in the CP cells of KO animals (data not shown). We postulatedthat many of the proteins increased in the CSF of KO mice representundigested substrates of NEU1, which are secreted extracellularly viaexacerbated LEX. Notably, multiple proteins differentially regulated inthe Neu1^(−/−) CSF have also been identified as possibledementia-predicting biomarkers associated with Alzheimer's disease (FIG.2). Thus, profound alterations of cellular physiology in one normallyNeu1-rich brain region may cause subsequent downstream changes that arehighly suggestive of an Alzheimer's-like status when Neu1 is lost. Forthis reason, we hypothesized that the observed changes in composition ofthe Neu1^(−/−) CSF would be paralleled by altered characteristics ofneural cells in the brain parenchyma. We were intrigued by theobservation that the other area of the WT brain expressing Neu1 at highlevels is the hippocampus (data not shown), one of the most intenselystudied structures of the brain in the AD field. In agreement with ourdefining paradigm of Neu1 reduction resulting in Lamp-1 accumulation andsubsequent excessive LEX, we first looked at Lamp1 and observed a markedincrease of this protein throughout the KO brain (data not shown). Thiswas confirmed by immunoblot analysis of brain hippocampal proteinextracts that identified increased amount of an oversialylated Lamp-1(data not shown). Based on these results, we hypothesized that cells inthe brain parenchyma of Neu1^(−/−) mice could also exert excessive LEX.Remarkably, Lamp1 was highly expressed in the microglia population(F4/80 staining, data not shown) suggesting that this cell populationmight be the most exocytic in the brain parenchyma, as previouslydemonstrated for BM macrophages. We investigated this by culturing WTand KO microglia. We tested their exocytic activity by measuring thelevels of active lysosomal hydrolases present in the medium, and found amarked increase of active lysosomal β-hexosaminidase in the medium fromKO microglia (data not shown).

We next examined the histopathological characteristics of the KOhippocampus and noticed numerous, abnormal eosinophilic bodies (data notshown). They were variable in size and mostly contained granularproteinaceous material (data not shown). At the EM level, these bodieswere identified as swollen dystrophic neurites containing numerousvacuoles of abnormal morphology resembling autophagic vacuoles (data notshown). These features were highly reminiscent of the distendeddystrophic neurites associated with AD. Therefore, we began a fullcharacterization of these structures, starting with antibodies reactiveto the N-terminal portion of APP and found a time-dependent, progressiveaccumulation of this protein in the pyramidal neurons of the thirdsubregion of the Cornus ammonis (CA3) of the hippocampus of Neu1^(−/−)brain (data not shown). To test if this phenotype was directly linked tothe Neu1 deficiency, we analyzed the sialylation status of APP in theNeu1^(−/−) brain. Evidence for oversialylation of APP came fromimmunoprecipitation studies; equal amount of hippocampal proteinextracts from wild-type and Neu1^(−/−) brain samples wereimmunoprecipitated with an APP C-terminal antibody and were examinedwith sambucus nigra lectin (SNA) that binds preferentially to sialicacid attached to terminal galactose with (α-2,6) linkages (data notshown).

It is well established that a slight overexpression of APP is a riskfactor for the development of AD and duplication of the APP locus infamilial AD and Down syndrome patients is the basis of early onset AD.We therefore tested a number of canonical histological markers commonlyapplied for the diagnosis of AD in the brain of Neu1^(−/−) mice. Swollendystrophic neurites were readily detected with thioflavin S fluorescencesuggesting they were structurally close to amyloid deposits (data notshown). Modified Bielschowsky silver stain also highlighted scatteredsilver-positive neuritic structures (data not shown), not found in agedmatched WT mice. The APP accumulating neurites were also immunoreactivewith antibodies recognizing APP/Aβ (data not shown), and were positivewhen stained with an antibody against the β-amyloid isoform ending atthe 42nd amino acid (A1342) (data not shown). Most importantly, almostall the APP+ dystrophic neurites were immunostained with ubiquitin,neurofilaments and tau antibodies indicating the presence of proteinaggregates and extensive cytoskeletal abnormalities in these structures(data not shown). We believe that APP accumulation in these dystrophicneurites contributes to the formation of toxic amyloid peptides (Aβ)because quantitative determination of Aβ40 and Aβ42(43) showed elevatedAβ peptides in the Neu1^(−/−) brain (data not shown). Based on thesedata, we conclude that Neu1 deficiency is directly linked to earlypathogenic events observed in AD.

The APP+ neurites may represent early events in the pathogenesis of theneuropil threads characterized by true amyloid and plaque deposition.APP overexpression in neurons or neurites may be toxic and causedegeneration with release of this protein. Exocytic microglia could thencontribute to the pathogenic process by releasing into the extracellularspace activated lysosomal enzymes that progressively process the APPproducing toxic Aβ peptides.

Here we have identified a novel mechanism orchestrated by deficiency oflysosomal Neu1 that promotes deposition of oversialylated APP which inturn may constitute a risk factor for the development of late-onsetnon-familiar AD.

The discovery of novel putative Neu1 substrates, including APP, and theoccurrence of deregulated LEX in the brain/CSF could provide a novel setof biomarkers for the diagnosis of AD and set the stage for innovativetherapeutic approaches to prevent/modulate APP/Aβ formation.

Example 4: Metabolic Control of Chemotherapy Resistance and MetastasisSummary

The dual dangers of cancer progression are chemotherapy resistance andmetastatic growth, both of which depend on the metabolic status of tumorcells. Here, we demonstrate that the lysosomal sialidase NEU1 plays adefining role in the development of both phenotypes by negativelyregulating the physiological process of lysosomal exocytosis. Cancercells use this mechanism to sequester and purge lysosomotropicchemotherapeutics, thereby developing drug resistance. Moreover,exocytosed active lysosomal enzymes from tumors degrade theextracellular matrix of surrounding tissue, compromising its ability tocontain tumor spread. Tumor-prone mice haploinsufficient for Neu1develop highly aggressive rare forms of cancer, confirming a role forNEU1 in controlling malignancy. In addition, downregulation of NEU1 iscommon in multiple human cancers. We propose that NEU1 functions as abona fide tumor suppressor by restraining lysosomal exocytosis in cancercells, precluding the development of a drug resistant and invasivephenotype.

Highlights

-   -   Lysosomal sialidase NEU1 negatively regulates lysosomal        exocytosis in cancer cells    -   Increased lysosomal exocytosis confers chemotherapy resistance        and invasiveness    -   Neu1 haploinsufficiency potentiates tumor growth and spread in        Arf^(−/−) mice    -   Downregulation of NEU1 is observed in multiple human cancers

INTRODUCTION

The lysosomal glycosidase N-acetyl-α-neuraminidase 1 (NEU1) is the mostabundant and widely expressed mammalian sialidase. Its canonicalfunction is to remove α2,6- or α2,3-linked terminal sialic acids fromthe saccharide chains of glycoproteins, glycolipids (gangliosides),oligosaccharides, and polysaccharides (Monti et al., 2010). Geneticdeficiency of NEU1 results in impaired catabolism of sialic acids on itstarget substrates, which in turn, affects countless cellular functionsand leads to the loss of cell and tissue homeostasis. The pathogeniceffects of NEU1 loss of function are obvious in the lysosomal storagedisease sialidosis, a severe neurosomatic condition in children andadolescents that affects most of the systemic organs and the nervoussystem (d'Azzo, 2009; Thomas, 2001).

In cancer, altered sialylation of glycoconjugates at surface membranesis considered a central determinant of the neoplastic process, though itis often unclear how changes in glycan composition result in aberrantbiological outcome (Varki et al., 2009; Wang, 2005). This dynamicposttranslational modification involving a charged sugar moiety cangreatly modify the biochemical and functional properties of proteins andlipids, thereby affecting cell-cell and cell-extracellular matrix (ECM)interactions, cell migration and adhesion patterns, intracellularsignaling and metastatic potential (Varki et al., 2009; Wang, 2005;Hedlund et al., 2008; Uemura et al., 2009). Excessive sialic acidcontent can result from two opposing processes, upregulation of thesynthetic enzymes sialyltransferases that control the regulated transferof sialic acids to nascent oligosaccharide moieties; and loss ofactivity of the sialidases that affect the same sugar nucleotidelinkages. For example, a compelling recent study has implicated theoverexpression of the sialyltransferase ST6GalNAc-V in the enhancedmetastatic potential of breast cancer cells, most likely throughabnormal sialylation of as yet unidentified proteins (Bos et al., 2009).One could argue that loss or downregulation of NEU1 would necessarilyresult in abnormal processing of sialylated substrates, making thecatabolic arm of this post-translational modification as relevant forcancer progression and growth. In fact, changes in the expression levelsof sialidases have been associated with cell migration and metastasis(Kato et al., 2001; Miyagi et al., 1994; Sawada et al., 2002; Uemura etal., 2009).

A previously unknown function for the sialidase NEU1 with greatrelevance to cancer has recently been discovered: that of negativeregulator of lysosomal exocytosis (LEX) (Yogalingam et al., 2008). Thisubiquitous, calcium-regulated physiological process entails therecruitment to the cytoskeletal network of a selective pool of lysosomesthat dock at the plasma membrane (PM); their limiting membrane thenfuses with the PM in response to calcium influx and their luminalcontent is released extracellularly (Bossi and Griffiths, 2005; Andrews,2000; Rodriguez et al., 1997). NEU1's function in this process ismediated via the lysosomal associated membrane protein 1 (LAMP1), anatural substrate of NEU1 (Yogalingam et al., 2008), which waspreviously implicated in the peripheral movement of lysosomes (Reddy etal., 2001). LAMP1 is a heavily glycosylated and sialylated structuralcomponent of the lysosomal membrane whose regulated turnover isconsiderably delayed when the protein is oversialylated due to loss ofNEU1 activity (Yogalingam et al., 2008). Long-lived oversialylated LAMP1changes the lysosomal membrane topology and trafficking, therebyincreasing the number of exocytic lysosomes docked at the PM ready tofuse and secrete their contents (Yogalingam et al., 2008).

Recent reports have made explicit calls for more attention to the areaof regulated exocytosis and cancer (Chan and Weber, 2002; Hendrix etal., 2010; Palmer et al., 2002). The involvement of this process as amediator of malignant growth and invasiveness has been postulated inview of the deregulated activities of vesicular trafficking effectorsobserved in some cancers (Hendrix et al., 2010; Palmer et al., 2002).Exocytosis can impact at least two critical areas of malignantprogression: abnormal remodeling of the ECM and promoting the efflux ofchemotherapeutic agents, many of which are lysosomotropic.Anthracyclines, cisplatin, and sunitinib are examples of such drugs thathave been directly visualized in lysosomes, whose trafficking couldgreatly influence the intracellular exposure to the drug (Gotink et al.,2011; Hurwitz et al., 1997; Safaei et al., 2005). The degradation andremodeling of the ECM has been attributed to the presence of lysosomalproteases in the extracellular environment and has been stronglycorrelated with tumor invasiveness and metastasis (Khan et al., 1998a;Khan et al., 1998b; Matarrese et al.).

In the present study we demonstrate that downregulation of NEU1 activitylevels in invasive cancer results in the accumulation of anoversialylated LAMP1 and in enhanced lysosomal exocytosis. Thecombination of these processes initiates a cascade of events that favortumor progression, invasiveness and resistance to chemotherapeuticdrugs. These results establish that NEU1 functions as a tumor suppressorby negatively regulating lysosomal exocytosis.

Results NEU1 Expression is Inversely Related to Lysosomal Exocytosis inRhabdomyosarcoma Cell Lines

Rhabdomyosarcoma (RMS), which arises in skeletal muscle, is the mostcommon soft-tissue malignancy in children and adolescents (Ognjanovic etal., 2009). This cancer is classified into two subtypes, embryonal RMS,which typically has a favorable prognosis, and alveolar RMS, which isassociated with poor prognosis (Ognjanovic et al., 2009). RMS is treatedprimarily with surgical resection and chemotherapy; common complicationsare metastases and chemotherapy resistance, both of which could resultfrom excessive LEX.

To investigate the role of NEU1 in regulating LEX in cancer cells, wechose two human RMS cell lines that express different amounts of NEU1.RH41 and RH30 cells were both derived from alveolar RMS tumors (Houghtonet al., 2007). Affymetrix mRNA microarray analysis of NEU1 expressionshowed that RH41 cells express a relatively high level of NEU1 mRNAcompared with that expressed by RH30 cells. We confirmed this findingusing semiquantitative and real-time PCR (data not shown). The mRNAresults correlated well with the levels of NEU1 activity (data notshown). Immunofluorescent labeling also revealed a typical lysosomaldistribution of NEU1 in both cell lines, but expression was markedlyhigher in the RH41 cells (data not shown).

On the basis of the different patterns of NEU1 expression in the RMScell lines, we characterized their LEX profiles. Western blot analysisof LAMP1 confirmed that the protein was more abundant in the low-NEU1RH30 cells and had a higher molecular weight, which was indicative ofincreased sialic acid content (data not shown). In addition, usingconfocal immunofluorescence, we observed a LAMP1⁺ signal on the cellsurface of nonpermeabilized RH30 cells but not on RH41 cells. Thissuggests an enhanced tendency of a LAMP1-marked pool of lysosomes in thelow-NEU1 cells to dock at and fuse with the PM, leading to accumulationof LAMP1 at that site (data not shown). We further monitored lysosomaltrafficking in real time by capturing confocal images of lysotrackerred-tagged puncta. In RH30 cells, lysosomes were preferentially capturedat the cell periphery and continuously dispatched from the cell centeroutward; in contrast, RH41 cells had virtually no lysosomes residingoutside the perinuclear space (data not shown). Total internalreflection (TIRF) microscopic analysis confirmed that the peripherallysosomes in RH30 cells were in close proximity to the PM. Live RH30cells stained with lysotracker green showed evidence of signallocalization within the range of TIRF-sensing, but RH41 cells did not(data not shown). Finally, we measured the extracellular activity of thelysosomal enzyme β-hexosaminidase (β-Hex) in culture medium from eachcell line as a measure of released lysosomal content. RH30-conditionedmedium contained considerably more β-Hex than did RH41-conditionedmedium, in inverse relation with their respective levels of NEU1 (datanot shown).

NEU1 Expression Levels Influence the Extent of Lysosomal Exocytosis

To pinpoint the primary role of NEU1 in the exocytic phenotype, weengineered stable NEU1-modified clones of the 2 RMS cell lines by usingretroviral vectors. Modified RH30 cells overexpressing NEU1(RH30^(NEU1)) and RH41 cells with silenced NEU1 (RH41^(shNEU1)) werefirst characterized for their NEU1 protein levels and activity toconfirm the successful reversion of their NEU1 expression patterns withrespect to the corresponding empty-vector controls (data not shown). LEXwas then measured in both modified cell lines and corresponding controlsusing 3 parameters: LAMP1 levels, TIRF analysis, and enzyme activitiesin conditioned media. LAMP1 levels were inversely proportional to thelevels of NEU1 activity in the modified lines (data not shown). Inaddition, LAMP1⁺ immunofluorescence was notably localized to the cellperiphery of RH41^(shNEU1) cells and control RH30^(empty) cells comparedto their high-NEU1 counterparts (RH30^(NEU1); RH41^(empty)) (data notshown). TIRF imaging confirmed the peripheral trafficking of lysosomesand their tendency to cluster at the termini of cell extensions in theRH41^(shNEU1) cells (data not shown). In contrast, this feature was lostin RH30^(NEU1) cells (data not shown).

Finally, the activity of lysosomal β-Hex was assayed in the culturemedium from each modified cell line and control. The media from thelow-NEU1 cells contained significantly more β-Hex activity (data notshown). Together, these results demonstrate that the NEU1 status of acell is sufficient to determine its LEX phenotype.

Increased Lysosomal Exocytosis Correlates with Doxorubicin Resistance

We next looked at the functional ramifications of NEU1 control over LEXin relation to the response of the parental cell lines tochemotherapeutic drugs. RH41 and RH30 cells have been shown to respondto a variety of antineoplastic therapy in markedly different way(Houghton et al., 2007; Petak et al., 2000). We found that upon exposureto doxorubicin (DOXO), RH41 cells readily apoptosed; RH30 cells wereresistant to treatment (data not shown). To link these phenotypes to thelevel of NEU1 activity in these cells, we first confirmed that DOXO wasconcentrated in their lysosomes. The native red fluorescence of the drugcolocalized with lysotracker green, revealing that DOXO-loaded lysosomeswere present in both parental RMS cell lines, albeit differentlydistributed throughout the cytoplasm (data not shown). Specifically,after 2 hours of treatment, DOXO-loaded lysosomes in RH41 cellsclustered in the perinuclear region, but those in RH30 cells did not(data not shown). Overnight live imaging of lysosome trafficking uponDOXO exposure confirmed these trends (data not shown). The intracellularvisualization of DOXO at 4 hours confirmed that the drug was primarilyconcentrated in the nuclei of RH41 cells, while in the RH30 cells afraction of the drug remained lysosomal (data not shown).

The increase in LEX observed in the low-NEU1 RH30 cells could promotethe efflux of DOXO, hence making the cells insensitive to treatment.This prediction was further supported by the fact that these cells donot express the multidrug-resistance protein 1 (p-glycoprotein 1) thatfunctions in a well-known cellular mechanism for evading drug toxicity(Cocker et al., 2000). By capturing and quantifying the effluxed redfluorescence from the RH30 culture medium, we showed that DOXO wasindeed released from these cells (data not shown).

Finally, we generated DOXO dose response curves in both parental celllines and the modified lines to examine the differences in theirapoptotic responses. Immunoblots of the cleaved poly (ADP-ribose)polymerase (PARP), a canonical apoptotic marker, were used for thispurpose (data not shown). The RH41^(shNEU1) cells were more resistant toapoptosis than were the corresponding unmodified cells (data not shown).In contrast, the RH30^(NEU1) cells were more sensitive to DOXO than weretheir unmodified controls (data not shown). In view of these results, wetested whether inhibiting LEX with the calcium channel blocker verapamilwould sensitize the parental RH30 cells.

Verapamil is a p-glycoprotein inhibitor, but it also inhibits drugefflux in the absence of p-glycoprotein, which suggests an alternativeefflux mechanism (Chiu et al., 2010). Because LEX depends on calciuminflux, we hypothesized that this commonly used calcium channel blockerwould inhibit this process. Upon co-treatment of the RH30 cells withverapamil and DOXO, the lysosomes accumulated the drug and clustered inthe perinuclear region (data not shown). The cells then underwentapoptosis, as measured by PARP cleavage and morphological analysis (datanot shown).

NEU1-Dependent Exacerbation of Lysosomal Exocytosis Increases theInvasive Capacity of Cancer Cells

The second feature of cancer cells that we predicted would be affectedby excessive LEX is invasiveness, because degradation of the ECM wouldcompromise the tissue's ability to contain the tumor. The release ofactive lysosomal resident proteases, particularly cathepsin B,correlates with basement membrane perforation and metastasis (Khan etal., 1998a; Khan et al., 1998b; Matarrese et al.).

We determined that NEU1 levels, and in turn extent of LEX, in tumorcells are linked to their invasive properties. For this purpose, we usedthe parental RH41 and RH30 lines as representative of high- and low-NEU1cells, respectively. The ex vivo invasive potentials of these cells weremeasured using denucleated peritoneal basement membranes (Marshall etal., 2011) obtained from either wild-type or Neu1-knockout mice (datanot shown). Because the Neu1-knockout mouse is a model of constitutive,excessive LEX, its tissues and ECM already have been subjected toprogressive environmental stresses that mimic tumor-adjacent tissue(Yogalingam et al., 2008; Zanoteli et al., 2010). Compared to high-NEU1RH41, the low-NEU1 RH30 line more successfully invaded the wild-typesubstrate (data not shown). However, the peritonea from Neu1-knockoutmice were significantly more vulnerable to invasion from either RMS cellline than were the wild-type peritonea (data not shown). Notably, thehigh-NEU1 RH41 cells seeded on a Neu1-knockout peritoneum were asinvasive as the RH30 cells on a wild-type peritoneum, suggesting thatintrinsic LEX had conditioned the otherwise healthy tissue for cancerousinvasion (data not shown). The invasive properties of the RMS cell lineswere further evaluated in the Neu1-knockout peritoneum sections byimmunohistochemical visualization of the basement membrane components,laminin and collagen IV, both of which are cathepsin B substrates (Bucket al., 1992). Tissue exposed to RH30 cells underwent moredestruction/remodeling than did tissue exposed to RH41 cells (data notshown).

The inverse relationship between NEU1 activity and invasive potential ofthe RMS cells suggested that this is a general mechanism that may beused by other cancers. We, therefore, characterized other tumor celllines, in terms of their invasive potential and NEU1 status. We chosethe Ewing sarcoma cell lines, EW8 and SKNEP1, for their divergent NEU1levels. Similar to the RMS cell lines, the low-NEU1 SKNEP1 cellsaccumulated oversialylated LAMP1, were resistant to DOXO, and moresuccessfully invaded matrigel and peritoneal basement membranes than didthe high-NEU1 DOXO-sensitive EW8 cells (data not shown). We alsoanalyzed the colon carcinoma cell lines, SW480 and SW620, because oftheir well-characterized derivation (Leibovitz et al., 1976): SW480cells are from a primary colon carcinoma; SW620 cells are from ametastatic recurrence. The latter cells were also shown to be moreresistant to anti-neoplastic drugs than SW480 cells (Walker et al.,2010), and we found that they downregulated NEU1 activity. This was alsoparalleled by a drastic increase in LAMP1 levels compared to that inSW480 (data not shown).

The NEU1 Status of RMS Cells is the Primary Determinant of Differencesin their Invasive Capacity

To determine how NEU1 levels influence the invasiveness of RMS cells, weseeded the RH41^(shNEU1) and RH30^(NEU1) cells, along with theempty-vector controls, on a matrigel substrate consisting primarily oflaminin and collagen IV. After the cells were maintained in culture for2 days, the matrigel plugs were fixed and processed to visualize theingress of cells into the substrate. The cells with low-NEU1 activity(RH41^(shNEU1) and RH30^(empty)) successfully invaded the matrigel,whereas those with high-NEU1 activity did not (data not shown). Theseresults established that the NEU1 status of these cancer cells issufficient to predict their ability to invade a standardized substrate.

Neu1-Deficient Mice Generate More Aggressive Cancers in a Tumor-ProneModel

Using Neu1-heterozygous mice crossed into a tumor-prone model, theArf-knockout mouse (Kamijo et al., 1999), we tested whether excessiveLEX affects malignant invasion in vivo. Two groups of mice were comparedfor tumor outcome. The first group, Arf^(−/−)/Neu1^(+/+) mice, produceda range of tumors that reflected the type of neoplasms previouslyreported in Arf single knockouts (Kamijo et al., 1999). Typical tumorsin Arf-knockout mice younger than 10 months are poorly differentiatedsarcomas, though gliomas, lymphomas, and carcinomas have been observedin low frequency (Kamijo et al., 1999). Tumors in these mice arose atthe average age of 9 months and were typically focal (data not shown).The second group consisted of mice with an Arf^(−/−)/Neu1^(+/+)genotype. We anticipated that the low-Neu1 activity in these mice wouldencourage faster, more virulent growth of any developing tumors. Thiswas indeed the case; the mice developed tumors at an average age of 6months, which was significantly earlier than that seen in the Arf singleknockouts (p=0.029). In addition, tumor growth was so aggressive thatmice were quickly rendered moribund. These tumors were often locallyinvasive and achieved large volumes in a short time span. In one case,the tumor was fulminantly metastatic (data not shown). Tumors in theNeu1-heterozygous mice were sometimes pleomorphic, not resembling thosecommonly seen in genetically-engineered mice. Two such malignancies hadmorphologic and immunohistochemical features of the rhabdoid/epithelioidsarcoma-like group of tumors. They contained rhabdoid-type cellspositive for both vimentin and cytokeratin 8, cytoskeletal markers formesenchymal and epithelial cells, respectively (data not shown). Thisgroup of tumors has not, to our knowledge, been reported in mice, and inhumans is associated with aggressive biological behavior and a poorprognosis (Oda and Tsuneyoshi, 2006).

One additional small cohort of mice, Arf^(−/−)/Neu1^(−/−) doubleknockouts, was generated during this breeding program. Very few mice ofthis genotype were born (17 of 122 total mice), and the mice succumbedto sialidosis-related mortality early in life. For these reasons, we didnot include this cohort in the statistical analysis, though we reportthe outcomes of the few mice that developed tumors, one metastatic,before the sialidosis became fatal (data not shown).

The easily characterized tumors that arose in the Arf^(−/−)/Neu1^(+/+)mice provided an opportunity to investigate the expression of Neu1 intumors compared to their cells of origin. We observed a loss of Neu1immunostaining in tumors compared to that in normal tissue from the samemouse. This difference was seen in a carcinoma and a sarcoma (data notshown), supporting the notion that downregulation of Neu1 offersselective advantages to tumor growth.

Loss of NEU1 in Human Cancer

We reasoned that the pattern of Neu1 expression in theArf^(−/−)/Neu1^(+/+) mice might be reproduced in patients with cancer,who should have normal NEU1 activity. We first probed human RMS tissuesamples of the more common embryonic subtype for the levels of NEU1compared to healthy skeletal muscle controls. NEU1 was, in fact,downregulated in 10 of 12 (83.3%) RMS samples (data not shown). Four ofthe 10 showed a complete absence of NEU1 staining (data not shown). Tofurther characterize the physiological impact of NEU1 downregulation, weassessed the levels of its substrate LAMP1. LAMP1 immunohistochemistryrevealed a strong upregulation of this protein in 7 of 12 (58.3%)samples, including all 4 of those with complete loss of NEU1 (data notshown). We next decided to repeat this analysis on pancreatic ductaladenocarinoma samples to gauge how common NEU1 downregulation is acrosscancer types. Again, 10 of 12 (83.3%) samples showed a loss of NEU1compared to the originating ductal cells (data not shown). LAMP1staining was also performed and replicated the results seen in RMS, with7 of 12 (58.3%) carcinoma samples showing upregulation (data not shown).Collectively, these results suggest that downregulation of NEU1 is acommonly employed strategy in cancer cells, often coupled toaccumulation of LAMP1 and concomitant exacerbation of LEX.

Discussion

In this study we provide evidence that the downregulation of NEU1 andconsequent enhanced LEX imparts at least two crucial advantages tocancer cells: resistance to lysosomotropic chemotherapeutic agents andthe ability to become expansive and infiltrative. These findings suggesta tumor-suppressor function for NEU1.

The regulated expression of this pleiotropic lysosomal enzyme affectstwo cellular processes, sialylation of glycoconjugates and the extentand type of lysosomal trafficking, both of which have been consistentlyinvoked to explain tumor cell behavior in vivo (Hendrix et al., 2010;Varki, 2009). Thus far, oversialylation has been largely attributed toupregulation of the biosynthetic enzymes sialyltransferases (Dall'Olioand Chiricolo, 2001). For instance, ST6GalNAc-I[(α-N-acetyl-neuraminyl-2,3-β-galactosyl-1,3)-N-acetylgalactosaminideα-2,6-sialyltransferase-I] is thought to contribute to the generation ofthe mucin-associated sialyl-Tn antigen, a marker of metastatic carcinoma(Heimburg-Molinaro et al., 2011; Marcos et al., 2011), and ST6GalNAc-Vimparts metastatic potential to breast cancer (Bos et al., 2009). Bothof these sialyltransferases catalyze the transfer of sialic acids inα2,6-linkage to GalNAc (N-acetyl-galactosamine) residues found inglycoproteins and glycolipids, a process that may be readily reversed byNEU1. We propose that diminished or deficient NEU1 activity impinges oncancer cells in a manner that is equivalent to sialyltransferaseoverexpression, thereby causing impaired sialic acid catabolism oftarget NEU1 substrates. It is noteworthy in this respect that a query ofthe Oncomine database (Finak Breast www.oncomine.org/resource/main.html)revealed a substantial upregulation (p<0.01) of at least 3α2,6-sialyltransferases, including the ST6GalNAc-I enzyme, as well asST6GalNAc-III and ST6Gal-II. We found that these inversely correlatedwith NEU1 expression, which was itself significantly downregulated(p<0.01). The opposing expression levels of these enzymes in cancercould cause an underappreciated double insult on the cellular regulationof sialylation.

Our study identified over-sialylated LAMP1 as a critical regulator ofLEX in cancer, providing both a marker for excessive LEX and a possibletarget for inhibiting this process. Deregulation of other proteinsinvolved in vesicular trafficking has been observed in some cancers.Namely, upregulation of two LEX effectors, Rab27B and BAIAP3, was shownto enhance the metastatic growth and cell proliferation of breast cancerand desmoplastic small round cell tumors, respectively (Hendrix et al.,2010; Palmer et al., 2002), suggesting the involvement of an exocyticmechanism in neoplasia (Chan and Weber, 2002). Here we present evidenceto definitively link LEX to the important cancer phenotypes ofinvasiveness and drug resistance.

The suggestion that lysosomal trafficking influences multidrugresistance has been proposed, with some reviews explicitly calling formore research in this area (Castino et al., 2003; Groth-Pedersen, 2010).An early report noted that DOXO is concentrated into acidic vesiclesthat could exocytose their contents (Klohs and Steinkampf, 1988). Later,vinblastine was observed to specifically accumulate in lysosomes and beeffluxed in conjunction with lysosomal enzymes (Warren et al., 1991).The study on how chemotherapeutics are released from tumor cells haslargely focused on the upregulation of p-glycoprotein or other ABCtransporters (Coley, 2010). However, specific inhibitors of these pumpshave often proven unsuccessful in clinical trials, suggesting that othermechanisms of efflux are involved (Broxterman et al., 2009; Coley,2010). Again, our search of the Oncomine database revealed supportiveevidence for NEU1 mediating an important resistance mechanism. Whilep-glycoprotein expression was not significantly different (p=0.81)between responders and non-responders to the 5-FU and the topoisomerasepoison irinotecan in a data set for metastatic colon cancer, NEU1expression was strongly downregulated (p=0.0064) in the resistantpatients (Graudens Colon data set at the website located atoncomine.org/resource/main). Here we have established that theDOXO-resistant RMS cell line effluxes DOXO, despite lacking thep-glycoprotein. The same cells can then be made more sensitive to DOXOby inhibiting LEX, either by upregulating NEU1 or using the calciumchannel blocker verapamil. Thus, lysosomotropic drug efflux is under thecontrol of NEU1, as a critical determinant of LEX.

NEU1-regulated LEX also has a direct influence on theinvasive/metastatic potential of cancer cells. First, otherwise healthytissue subjected to excessive LEX, as in the case of Neu1-knockout mice,is more susceptible to cancer cell invasion. Second, the level of NEU1in cancer cells is inversely related to their invasive potential. Third,the combined loss of NEU1 in the tumor and the surrounding tissue invivo creates the conditions for highly aggressive cancers. TheArf^(−/−)/Neu1^(+/−) mice developed various tumors, including highlyaggressive, rare rhabdoid/epithelioid-like sarcomas of uncertainhistogenesis. Because no mouse model has been identified for this typeof cancer, the spontaneous generation of these tumors offers a possiblewindow of opportunity into the study of these rare and deadlymalignancies.

In addition to low-NEU1 status being a potential risk factor in thedevelopment of cancer, neoplastic transformation may place selectivepressure on tumor cells to downregulate NEU1, as demonstrated by theloss of Neu1 expression in the tumors developed by Arf single-knockoutmice. NEU1 activity levels in the normal human population range widely,with as-yet-unknown ramifications. It is predictable that individualswith low NEU1 activity, i.e., those with the type I sialidosis, would bemore vulnerable to cancer than are healthy controls. This appears to bethe case; Yagi and colleagues recently reported different neoplasticmalignancies in three siblings with type I sialidosis and suggested alink between these occurrences and NEU1 deficiency (Yagi et al., 2011).

We show here that NEU1 is robustly downregulated in malignant tissuesfrom human RMS and pancreatic adenocarcinoma compared to unmatchedhealthy tissue. Whether this is due to a preexisting deficiency or to atransformation-related change is unknown and deserves further attention.In either case, reduced NEU1 expression may be an informative marker,both because of the consistency of its downregulation and because of thephysiological ramifications of its loss. Coupled to accumulation ofsubstrate, NEU1 loss could function as a predictor of excessive LEX andits important consequences of increased invasiveness and drug resistancein tumors. Such information may help to shape treatment decisions and tooptimize the rational design of new drug combination trials.

Experimental Procedures Animals

Neu1^(+/−) mice were bred with Arf^(−/−) mice (kindly provided by Dr.Charles Sherr). The colony was expanded for 1 year, during which timethe birth rates and tumor burdens were documented. Full necropsies wereperformed by the St. Jude Veterinary Pathology Core. All mouseexperiments were performed according to animal protocols approved by ourinstitutional Animal Care and Use Committee and NIH guidelines.

Cell Culture

The RMS cell lines RH41, RH30 and Ewing sarcoma cell lines EW8, andSKNEP-1 were kindly provided by Drs. Gerard Grosveld and AndrewDavidoff. These cell lines were characterized by the PediatricPreclinical Testing Program at St. Jude Children's Research Hospital(Houghton et al., 2007). SW480 and SW620 adenocarcinoma colon cancercell lines were obtained from ATCC. Cells were maintained in DMEM orRPMI (Invitrogen) media supplemented with Glutamax (Sigma), penicillinand streptomycin (Invitrogen), and 10% cosmic calf serum (Hyclone).Puromycin-resistant clones were selected and maintained in mediasupplemented with puromycin (2 μg/mL, Sigma).

Antibodies and Reagents

We used commercial antibodies: anti-LAMP1 (Sigma), anti-laminin (Sigma),anti-α/β tubulin and anti-cleaved PARP (Cell Signaling). Polyclonalanti-NEU1 antibody has been previously described (Bonten et al., 2004).Verapamil (50 Sigma) and DOXO (3 μg/mL, LKT Laboratories) were added tothe culture media. Lysotracker Green DND-26 and Lysotracker Red DND-99were obtained from Invitrogen and applied per the manufacturer'sinstructions.

Immunoblotting

Tissue and cell-pellet preparation, Western blotting, and data analyseswere performed as previously described (Zanoteli et al., 2010).

Immunohistochemistry and Fluorescent Microscopy

Immunohistochemical analyses of mouse tissue sections and microarrayanalyses of human tissue samples (US BioMax, Inc.) were performed aspreviously described (Yogalingam et al., 2008). The use of human tissuesamples was approved by our Institutional Review Board.

Enzyme Assays

NEU1 and β-Hex enzymatic activities were measured with the appropriatefluorimetric substrates (Sigma) and normalized to BCA proteinconcentrations (Pierce Biotechnology) as described previously(Yogalingam et al., 2008).

Stable Clone Cell Lines

RH30 cells were transduced with the pBABE-puro retroviral vector(AddGene), either unmodified or containing the human NEU1 cDNA.Transduced cells were selected with puromycin (2 μg/mL), according tothe predetermined sensitivity of the cell line, and were maintained in0.5 μg/mL selection medium. RH41 cells were transfected with a panel ofNEU1 shRNA plasmids (RHS4533-NM000434, Open Biosystems) or theaccompanying empty vector control. The cells were selected withpuromycin and maintained under selection medium.

Doxorubicin Efflux Assay

Cells were plated and maintained at 50% confluency. The next day, theywere treated with medium containing 3 μg/mL DOXO for 2 hours. Treatedcells were then washed 3 times with PBS and cultured in DOXO-free mediumfor an additional 2 hours, to allow efflux of the drug into freshmedium. The conditioned medium was then centrifuged at 1000 rpm (867×g)for 5 minutes and a 500 μL aliquot was spun through an ULTRAFREE-MC 0.1μm Eppendorf centrifugal filter (Millipore) to capture the effluxeddrug. The filter was then placed on a microscopic slide and images ofthe red fluorescence filter were taken on a Nikon C1 microscope.

Invasion Assays

Peritoneal sections were harvested from wild-type or Neu1-knockout miceof matched ages and mounted over transwell inserts (Fisher), aspreviously described (Marshall et al., 2011). A total of 250,000 RMScells per line were then overlaid onto the peritoneal preparation andkept in culture for 8 days.

In a separate set of experiments, 250,000 RMS cells per line were seededonto 200 matrigel in a transwell dish and maintained in culture for 2days, as previously described (Sabeh et al., 2009).

Statistics

Data are expressed as mean±standard deviation (SD) and were evaluatedusing the Student's t-test for unpaired samples. P-values less than 0.05were considered statistically significant.

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Example 5: Lysosomal Dysfunction and Excessive Lysosomal Exocytosis Leadto Alzheimer's-Like Amyloidogenesis Abstract

Lysosomal exocytosis is a regulated physiological process responsiblefor the controlled secretion of metabolites from specialized secretorycells and for the maintenance of plasma membrane homeostasis in mostcell types. The lysosomal sialidase NEU1 is a pivotal negative regulatorof this process; genetic ablation of Neu1 in the mouse model of thechildhood disease sialidosis leads to exacerbated release of lysosomalcontent extracellularly with deleterious effects for tissue andextracellular matrix integrity. Here we show that Neu1^(−/−) micedevelop pathological and molecular changes in the brain that arereminiscent of Alzheimer's disease (AD). The synergistic action ofexcessive lysosomal exocytosis of neuronal cells and lysosomalaccumulation of oversialylated Neu1 substrates, including the amyloidprecursor protein, contribute to the amyloidogenic cascade. In addition,Neu1 downregulation, in a known model of AD, accelerates theamyloidogenic process; conversely, up-regulation of Neu1 in the samemodel reduces amyloid deposition and plaques formation. These data mayexplain some of the pathological mechanisms of AD and offer newtherapeutic targets along a previously unknown pathway.

Introduction

Lysosomes are the major site of compartmentalized degradation ofglycoproteins, glycolipids, as well as aged organelles, and in thiscapacity they are pivotal for the maintenance of cell homeostasis.Downregulation or deficiency of any of the lysosomal constituents, whosecoordinated activities control overall lysosomal function, disrupts thebalance between synthesis and degradation with detrimental effects onmultiple tissues and organs. This is particularly true for brain, whichis exquisitely sensitive to metabolic changes. One of these fundamentallysosomal enzymes is the sialidase NEU1, which initiates the catabolismof a plethora of sialoglyconjugate substrates by removing their terminalsialic acids. Aside from its canonical degradative function, NEU1 wasrecently identified as the enzyme that regulates the physiologicalprocess of lysosomal exocytosis (LEX), a function that NEU1 exerts bycontrolling the sialic acid content of one of its target substrates, thelysosomal associated membrane protein, LAMP1. LEX is a Ca²⁺-dependentregulated mechanism present in virtually all cell types. It begins withthe recruitment of a subset of lysosomes along the cytoskeleton to theplasma membrane (PM), followed by their docking at the PM, and fusionwith the PM, which releases the lysosomal luminal content into theextracellular space. The docking step of the pathway is mediated byLAMP1. In absence of NEU1, a long-lived, oversialylated LAMP1 specifiesan increased number of lysosomes poised to dock at the PM and engage inLEX upon Ca²⁺ influx. The end result is the exacerbated release oflysosomal content extracellularly, which results in abnormal remodelingof the extracellular matrix (ECM) and changes in PM and ECM composition.We determined that many of the systemic abnormalities downstream of NEU1deficiency in the mouse model of sialidosis could be attributed toexcessive LEX, although the downstream effects of this phenotype mightvary depending on the physiological characteristics of the affectedtissue.

Here we wished to investigate whether deregulated LEX could contributeto the progressive, neuropathological manifestations of the Neu1^(−/−)mice, which reflect those in children with sialidosis.

Results:

We first examined the pattern of expression of Neu1 in the normal brainand demonstrated that the enzyme was widely distributed throughout theparenchyma, with the highest expression in the hippocampus (data notshown). In line with this expression pattern, Lamp1 accumulated in anoversialylated state (data not shown), a feature that correlated withexcessive LEX in other cells and tissues of the KO mice. Lamp1 wasparticularly abundant in activated microglia and in the pyramidalneurons of the Neu1^(−/−) hippocampus (data not shown), suggesting thatthese cells could exhibit excessive LEX. We tested this possibility bymeasuring the levels of active lysosomal enzymes in the medium ofprimary microglia and neurosphere cultures, isolated from Neu1^(−/−/ARF)and WT^(/ARF) mice to increase the number of pluripotent cells andenhance cell viability. Neurospheres from both genotypes had similarcell composition, but the activity of lysosomal β-hexosaminidase (β-hex)was significantly increased only in the media of the Neu1^(−/−)microglia and Neu1^(−/−/ARF) neurospheres (data not shown), confirmingthe occurrence in these cells of excessive LEX. Notably, the levels ofLEX were similar in WT and Neu1^(−/−) primary astrocytes (data notshown), hence we attributed the enhanced exocytic activity measured inthe Neu1 deficient neurospheres to the neuronal population of thesecultures.

We argued that the increased levels of LEX in Neu1^(−/−) neurons andmicroglia could dramatically affect the architecture and composition ofthe brain parenchyma. In fact, histopathological examination of theNeu1^(−/−) brain identified numerous, abnormal eosinophilic bodies,particularly abundant in the CA3 subregion of the hippocampus (data notshown). They were heterogeneous in size and shape, and mostly containedamorphous, granular proteinaceous material, closely resembling theamyloid. These bodies were positive for the histological markersthioflavin S and modified Bielschowsky silver stain (data not shown).Moreover, clusters of these deposits were detected specifically in theCA3 of the Neu1^(−/−) hippocampus by systemic injection of Methoxy-X04,a finding that confirmed the occurrence of an amyloidogenic processdownstream of Neu1 deficiency (data not shown). At the ultrastructurallevel, the amyloid deposits were identified as swollen dystrophicneurites containing numerous vesicles of abnormal morphology and content(data not shown). Combined these phenotypic alterations were reminiscentof those characteristic of Alzheimer's disease (AD). AD is considered adisease of protein aggregates whose composition consists primarily ofamyloid precursor protein (APP) abnormally processed into amyloidβ-peptides (Aβ) and other proteolytic fragments.

To characterize the amyloid in the brain of Neu1^(−/−) mice, we usedantibodies cross-reacting with full length APP and found a progressiveand time dependent accumulation of this protein in the pyramidal neuronsof the CA3 region (data not shown). Ubiquitin and neurofilamentsantibodies also immunostained most of the APP+ dystrophic neurites,suggesting extensive cytoskeletal abnormalities in these structures(data not shown). Accumulation of APP was confirmed by immunoblots ofhippocampal lysates that demonstrated a marked increase of this proteinin Neu1^(−/−) samples (data not shown). Because it is well establishedthat increased expression of APP in both AD and Down syndrome patientsrepresents a risk factor for the development of the disease, we inferredthat NEU1 loss of function could predispose to an AD-like phenotype.

APP is a type-I membrane glycoprotein, which is glycosylated andsialylated; changes in its glycan makeup have been linked to aberrantprocessing of the protein leading to increased production and secretionof toxic Aβ peptides. We hypothesized that APP could be a naturalsubstrate of Neu1 and, if so, would accumulate in an oversialylatedstate in absence of Neu1 activity. Indeed, analysis of APP in Neu1^(−/−)hippocampal lysates with sambucus nigra lectin (SNA) confirmed thepresence of excess amounts of α-2,6 linked sialic acids on the protein(data not shown). In vitro enzymatic removal of all N- and O-glycansreleased a core-APP protein that was identical in size in the Neu1^(−/−)and WT samples, indicating that APP conformational changes in theNeu1^(−/−) brain were due to impaired removal of its sialic acids (datanot shown). Accumulated APP was also detected in crude lysosomalfractions (CLF) isolated from the KO hippocampi, together with two othersubstrates of Neu1 (data not shown), Lamp1 and cathepsin B. Theseresults identify APP as a novel substrate of Neu1 that could be at leastin part cleaved in the lysosomal compartment.

A crucial step in the amyloidogenic processing of APP is the generationof carboxy terminal fragments (CTFs), which are subsequently cleavedinto ft-amyloid. We therefore assessed their levels in Neu1^(−/−)samples, as predictive measure of abnormal Aft processing. CTFs levelswere increased in both Neu1^(−/−) hippocampal samples and in CLFcompared to those in WT samples (data not shown). Furthermore, β-amyloidwas abnormally present in Neu1^(−/−) CLF (data not shown) suggestingthat oversialylated APP is processed in the lysosomal compartment. Theseobservations were further supported by the detection of elevated amountsof the amyloid peptide Aβ42 (Aβ) both in the culture media ofNeu1^(−/−/ARF) neurospheres, and in the KO cerebrospinal fluid (data notshown). To ascertain the role of LEX in this amyloidogenic process, wecultured Neu1^(−/−/ARF) and Neu1^(WT/ARF) neurospheres in presence of ahuman TAMRA-conjugated, fluorescent Aβ42 (T-Aβ). T-Aβ was readily takenup by the cells and rerouted to late endosomes/lysosomes (data notshown). This fraction of the internalized peptide was then traffickedfrom the lysosomes to the PM via LEX, as determined by live imaging oflysotracker-labeled lysosomes with total internal reflection microscopy(data not shown). By counting the number of lysotracker+ organellesproximal to the PM we showed that Neu1^(−/−/ARF) cells had significantlyhigher number of T-Aβ-containing lysosomes clustered at the PM (data notshown). Moreover, when both neurosphere cultures were maintained in T-Aβfree medium for 24 h following exposure to the peptide we were able tocapture the T-Aβ fluorescence released extracellularly, and consequentlywe measured increased amounts of T-Aβ exocytosed into the medium ofNeu1^(−/−/ARF) cells compared to WT cells (data not shown). Thus, Aβ isabnormally secreted in absence of Neu1 and is released via LEX.

Together these results suggest that Neu1 loss of function and consequentexacerbation of LEX are predisposing factors to β-amyloidogenesis. Tofurther verify this assumption, we analyzed the effects of Neu1 ablationon amyloid β levels and plaque formation in vivo by using a wellcharacterized transgenic model of AD (5XFAD) that we crossed into theNeu1^(−/−) background. We first tested the expression levels of Neu1 inthe 5XFAD mouse line by immunohistochemistry. We observed a markeddownregulation of the enzyme, especially noticeable in the hippocampus,which was accompanied by increased expression of Lamp1 (data not shown).We also measured reduced Neu1 enzyme activity in primary neurospheresisolated from the 5XFAD hippocampi (data not shown). To further theseobservations, we demonstrated that the levels of APP were increased inhippocampal lysates isolated from 5XFAD/Neu1^(−/−) compared to those in5XFAD/WT (data not shown). Moreover, APP processing in these miceresulted in the accumulation of amyloid-β (data not shown), a findingthat supports the idea that downregulation or loss of Neu1 in 5XFAD miceaccelerates the β-amyloidogenic process likely via deregulated LEX.

Finally, to test whether Neu1 deficiency (and excessive LEX) couldrepresent a therapeutic target to revert an AD-like phenotype, we soughtto exogenously increase Neu1 activity in the brain of 5XFAD mice. Thelatter could be achieved by augmenting the intracellular expression ofthe Neu1 chaperone Protective Protein Cathepsin A (PPCA). We thereforeperformed stereotactic injection of an adeno-associated virus containingboth human NEU1 and PPCA (AAVNEU1/AAVPPCA) into the hippocampal regionof the 5XFAD mice. This vector combination directed sustained expressionof the transgenes in vitro (data not shown). Four weeks after injection,high expression of both PPCA and NEU1 was detected in brain sections ofmice treated with the recombinant AAV vectors (data not shown).Remarkably, the abundant number of amyloid plaques seen in the untreated5XFAD mice was reduced by 44.3±6.2% in the AAV injected mice, comparedto the same line injected only with carrier solution (data not shown).Immunoblots of hippocampal lysates from the injected 5XFAD miceconfirmed that both APP and β-amyloid levels were reduced (data notshown).

Conclusions:

In conclusion our results reveal an unsuspected mechanism of controlover APP processing by a lysosomal enzyme. We propose a two-hit model toexplain the amyloidogenic process downstream of Neu1 loss of function:the accumulation of an oversialylated APP which is abnormally processed,followed by the release of APP end products via excessive LEX. In thisscenario the activated microglia with increased LEX could engage in afeed forward pathogenic cascade by releasing active lysosomal enzymesextracellularly that may cleave APP, producing toxic Aβ peptides. Neu1position in the amyloidogenic pathway and its role as central regulatorof LEX may be exploited for new potential therapeutic targets totreat/delay plaque deposition and amyloid formation in AD.

That which is claimed:
 1. A method of determining the prognosis for asubject with cancer, comprising the steps of a) providing a subjectprofile comprising a lysosomal sialidase activity profile comprising twoor more values from different lysosomal sialidase activity markers, aNEU1 substrate sialylation activity profile or a NEU1 level activityprofile from a tumor sample from said subject; b) providing acorresponding reference profile comprising a lysosomal sialidaseactivity profile comprising two or more values from different lysosomalsialidase activity markers, a NEU1 substrate sialylation activityprofile or a NEU1 level activity profile from a control sample, whereinthe subject profile and the reference profile comprise one or morevalues representing lysosomal sialidase activity, NEU1 substratesialylation activity or NEU1 level activity; and c) comparing saidsubject and said reference lysosomal sialidase activity profiles tothereby determine the prognosis for said subject with cancer, wherein alower lysosomal sialidase activity, a higher NEU1 substrate sialylationactivity or a higher NEU1 level activity of said subject as compared tothe lysosomal sialidase activity, NEU1 substrate sialylation activity orNEU1 level activity of said reference results in a prediction of aninvasive cancer for said subject.